Benzodiazepine derivatives

ABSTRACT

The invention relates to novel benzodiazepine derivatives with antiproliferative activity and more specifically to novel benzodiazepines of formula (I) and (II), in which the diazepine ring (B) is fused with a heterocyclic ring (CD), wherein the heterocyclic ring is bicyclic or a compound of formula (III), in which the diazepine ring (B) is fused with a heterocyclic ring (C), wherein the heterocyclic ring is monocyclic. The invention provides cytotoxic dimers of these compounds. The invention also provides conjugates of the monomers and the dimers. The invention further provides compositions and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the compounds or conjugates of the invention. The invention further relates to methods of using the compounds or conjugates for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/700,131, filed on Feb. 4, 2010, which claims the benefit of thefiling date, under 35 U.S.C. §119(e), of U.S. Provisional ApplicationNo. 61/150,201, filed on Feb. 5, 2009, the entire contents of each ofthe above-referenced applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel cytotoxic compounds and cytotoxicconjugates comprising these cytotoxic compounds and cell-binding agents.More specifically, this invention relates to novel benzodiazepinecompounds (e.g., indolinobenzodiazepines or oxazolidinobenzodiazepines),derivatives thereof, intermediates thereof, conjugates thereof, andpharmaceutically acceptable salts thereof, which are useful asmedicaments, in particular as antiproliferative agents.

BACKGROUND OF THE INVENTION

Benzodiazepine derivatives are useful compounds for treating variousdisorders, and include medicaments such as, antiepileptics(imidazo[2,1-b][1,3,5]benzothiadiazepines, U.S. Pat. No. 4,444,688; U.S.Pat. No. 4,062,852), antibacterials(pyrimido[1,2-c][1,3,5]benzothiadiazepines, GB 1476684), diuretics andhypotensives (pyrrolo(1,2-b)[1,2,5]benzothiadiazepine 5,5 dioxide, U.S.Pat. No. 3,506,646), hypolipidemics (WO 03091232), anti-depressants(U.S. Pat. No. 3,453,266); osteoporosis (JP 2138272).

Recently, it has been shown in animal tumor models that benzodiazepinederivatives, such as pyrrolobenzodiazepines (PBDs), act as anti-tumoragents (N-2-imidazolyl alkyl substituted1,2,5-benzothiadiazepine-1,1-dioxide, U.S. Pat. No. 6,156,746),benzo-pyrido or dipyrido thiadiazepine (WO 2004/069843),pyrrolo[1,2-b][1,2,5]benzothiadiazepines andpirrole[1,2-b][1,2,5]benzodiazepine derivatives (WO2007/015280),tomaymycin derivatives (e.g., pyrrolo[1,4]benzodiazepines), such asthose described in WO 00/12508, WO2005/085260, WO2007/085930, and EP2019104. Benzodiazepines are also known to affect cell growth anddifferentiation (Kamal A., et al., Bioorg Med Chem. 2008 Aug. 15;16(16):7804-10 (and references cited therein); Kumar R, Mini Rev MedChem. 2003 June; 3(4):323-39 (and references cited therein); Bednarski JJ, et al., 2004; Sutter A. P, et al., 2002; Blatt N B, et al., 2002),Kamal A. et al., Current Med. Chem., 2002; 2; 215-254, Wang J-J., J.Med. Chem., 2206; 49:1442-1449, Alley M. C. et al., Cancer Res. 2004;64:6700-6706, Pepper C. J., Cancer Res 2004; 74:6750-6755, Thurston D.E. and Bose D. S., Chem Rev 1994; 94:433-465; and Tozuka, Z., et al.,Journal of Antibiotics, (1983) 36; 1699-1708. General structure of PBDsis described in US Publication Number 20070072846. The PBDs differ inthe number, type and position of substituents, in both their aromatic Arings and pyrrolo C rings, and in the degree of saturation of the Cring. Their ability to form an adduct in the minor groove enables themto interfere with DNA processing, hence their potential for use asantiproliferative agents.

There still exists a need for novel benzodiazepine derivatives aseffective and safe therapeutics for treating a variety of proliferativedisease states, such as cancer.

SUMMARY OF THE INVENTION

One object of the invention is to provide novel benzodiazepines offormula (I) and (II), in which the diazepine ring (B) is fused with aheterocyclic ring (CD), wherein the heterocyclic ring is bicyclic,

wherein:the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H, or an amine protecting moiety that converts the compoundinto a prodrug that can be transformed into the free amine in vitro orin vivo;Y is selected from —OR, an ester represented by —OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxylamine represented by NR′R″, amide represented by —NRCOR′, apeptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand are selected from H, substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbon atoms apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000, a 5- or 6-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, a5 to 18 membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, a 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P wherein the substituent is selected from halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selectedfrom H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from1 to 10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), whereinn is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur. a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to6 heteroatoms selected from O, S, N and P and R₁₀ optionally is SR₁₃ orCOR₁₃, wherein R₁₃ is selected from linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, a 5-or 6-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, a 5 to 18membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, 3 to 18-membered heterocyclic ring having1 to 6 heteroatoms selected from O, S, N and P and R₁₁ can also be OR₁₄,wherein R₁₄ is H or has the same definition as R, optionally, R″ is anOH;W is C═O, C═S, CH₂, BH (B=Boron), SO or SO₂;R₁, R₂, R₃, R₄, are each independently selected from H, substituted orunsubstituted linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, or a substituentselected from a halogen, OR₇, NR₈R₉, NO₂, NRCOR′, SR₁0, a sulfoxiderepresented by SOR′, a sulfone represented by —SO₂R′, a sulfite —SO₃, abisulfite —OSO₃, a sulfonamide represented by SO₂NRR′, cyano, an azido,guanidinium [—NH(C═NH)NH₂], —COR₁₁, —OCOR₁₁ or —OCONR₁₁R₁₂ wherein R₇,R₈, R₉, R₁₀, R₁₁ and R₁₂ have the same definitions as given above,optionally, any one of R₁, R₂, R₃, R₄ is a linking group that enableslinkage to a cell binding agent via a covalent bond or is selected froma polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl,poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing alinking group that enables linkage to a cell binding agent;R₅ is selected from OR₁₅, CRR′OH, SH, CRR′SH, NHR₁₅ or CRR′NHR₁₅,wherein R₁₅ has the same definition as R., R and R′ have the samedefinition as given above; optionally, R₅ is a linking group thatenables linkage to a cell binding agent via a covalent bond or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent;R₆ is OR, SR, NRR′, wherein R and R′ have the same definition as givenabove, or optionally R₆ is a linking group;Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000; or their pharmaceutically acceptable solvates, salts, hydratesor hydrated salts, their optical isomers, racemates, diastereomers,enantiomers of these compounds.provided that the compound has no more than one linking group thatenables linkage to a cell binding agent via a covalent bond.

A second object of the invention is to provide novel benzodiazepines offormula (III), in which the diazepine ring (B) is fused with aheterocyclic ring (C), wherein the heterocyclic ring is monocyclic,

wherein:the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting moiety that converts the compoundinto a prodrug;Y is selected from —OR, an ester represented by —OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxylamine represented by NR′R″, amide represented by —NRCOR′, apeptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand selected from H, substituted or unsubstituted linear, branched orcyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000, a 5- or 6-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, a5 to 18 membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms 3 to18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O,S, N and P, wherein the substituent is selected from halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₃,wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selectedfrom H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from1 to 10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), whereinn is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to6 heteroatoms selected from O, S, N and P and R₁₀ optionally is SR₁₃ orCOR₁₃, herein R₁₃ is selected from linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, a 5-or 6-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, a 5 to 18membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P and R₁₁ can also be OR₁₄, wherein R₁₄ is H or has the samedefinition as R, optionally R″ is OH;W is C═O, C═S, CH₂, BH, SO or SO₂;R₅ is selected from OR₁₅, CRR′OH, SH, CRR′SH, NHR₁₅ or CRR′NHR₁₅,wherein R₁₅ has the same definition as R. or is a linking group thatenables linkage to a cell binding agent via a covalent bond or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent;R₆ is OR, SR or NRR′, wherein R and R′ have the same definition as givenabove, optionally R₆ is a linking group;X′ is CH₂, NR, CO, BH, SO or SO₂;Y′ is O, CH₂, NR or S;Z′ is CH₂ or (CH₂)_(n), wherein n is 2, 3 or 4; or theirpharmaceutically acceptable solvates, salts, hydrates or hydrated salts,their optical isomers, racemates, diastereomers, enantiomers or thepolymorphic crystalline structures of these compounds;provided that the compound has no more than one linking group thatenables linkage to a cell binding agent via a covalent bond.

A third object of the invention is to provide cytotoxic dimers (IV), (V)and (VI)

of the benzodiazepine monomers of formulas (I) and (II) and (III),respectively, in which the dimer compounds optionally bear a linkinggroup that allows for linkage to cell binding agents,

wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting moiety that converts the compoundinto a prodrug;

Y is selected from —OR, an ester represented by OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxylamine represented by NR′R″, amide represented by —NRCOR′, apeptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand are selected from H, substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to2000, a 5- or 6-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, a5 to 18 membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P wherein the substituent is selected from halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein of R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selectedfrom H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from1 to 10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), whereinn is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms, 3 to 10-membered heterocyclic ring having 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P and R₁₀ is optionally SR₁₃ or COR₁₃, wherein R₁₃ isselected from linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms, 3 to 18-membered heterocyclic ring having 1to 6 heteroatoms selected from O, S, N and P, optionally R₁₁ is OR₁₄,wherein R₁₄ has the same definition as R, optionally R″ is OH;W is C═O, C═S, CH₂, BH, SO or SO₂;R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently selectedfrom H, substituted or unsubstituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to 2000, or asubstituent selected from a halogen, guanidinium [—NH(C═NH)NH₂], OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined above optionally,any one of R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, or R₄′ is a linking group thatenables linkage to a cell binding agent via a covalent bond or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent,Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;R₆ is OR, SR or NRR′, wherein R and R′ have the same definition as givenabove, optionally R₆ is a linking group;X′ is selected from CH₂, NR, CO, BH, SO or SO₂ wherein R has the samedefinition as given above;Y′ is O, CH₂, NR or S, wherein R has the same definition as given above;Z′ is CH₂ or (CH₂)_(n), wherein n is 2, 3 or 4, provided that X′, Y′ andZ′ are not all CH₂ at the same time;A and A′ are the same or different and are selected from O, —CRR′O, S,—CRR′S, —NR₁₅ or CRR′NHR₁₅, wherein R and R′ have the same definition asgiven above and wherein R₁₅ has the same definition as R.D and D′ are same or different and independently selected from linear,branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms, optionally substituted with any one of halogen, OR₇, NR₈R₉, NO₂,NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfone represented by—SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamide represented bySO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂, wherein thedefinitions of R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined above, or apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;L is an optional phenyl group or 3 to 18-membered heterocyclic ringhaving 1 to 6 heteroatoms selected from O, S, N and P that is optionallysubstituted, wherein the substituent is a linking group that enableslinkage to a cell binding agent via a covalent bond, or is selected fromlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms, optionally substituted with any one of halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ have the same definitions as givenabove, a polyethylene glycol unit (—OCH₂CH₂)n, wherein n is an integerfrom 1 to 2000; optionally, L itself is a linking group that enableslinkage to a cell binding agent via a covalent bond; or theirpharmaceutically acceptable solvates, salts, hydrates or hydrated salts,their optical isomers, racemates, diastereomers, enantiomers or thepolymorphic crystalline structures of these compounds; provided that thecompound has no more than one linking group that enables linkage to acell binding agent via a covalent bond.

A fourth object of the invention is to provide conjugates of cellbinding agents with the novel benzodiazepine compounds or derivativesthereof of the present invention. These conjugates are useful astherapeutic agents, which are delivered specifically to target cells andare cytotoxic.

The present invention includes a composition (e.g., a pharmaceuticalcomposition) comprising novel benzodiazepine compounds, derivativesthereof, or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier). Thepresent invention also includes a composition (e.g., a pharmaceuticalcomposition) comprising novel benzodiazepine compounds, derivativesthereof, or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier), furthercomprising a second therapeutic agent. The present compositions areuseful for inhibiting abnormal cell growth or treating a proliferativedisorder in a mammal (e.g., human). The present compositions are alsouseful for treating depression, anxiety, stress, phobias, panic,dysphoria, psychiatric disorders, pain, and inflammatory diseases in amammal (e.g., human).

The present invention includes a method of inhibiting abnormal cellgrowth or treating a proliferative disorder in a mammal (e.g., human)comprising administering to said mammal a therapeutically effectiveamount of novel benzodiazepine compounds, derivatives thereof, orconjugates thereof, (and/or solvates and salts thereof) or a compositionthereof, alone or in combination with a second therapeutic agent.

The present invention includes a method of synthesizing and using novelbenzodiazepine compounds, derivatives thereof, and conjugates thereoffor in vitro, in situ, and in vivo diagnosis or treatment of mammaliancells, organisms, or associated pathological conditions.

The compounds of this invention, derivatives thereof, or conjugatesthereof, and compositions comprising them, are useful for treating orlessening the severity of disorders, such as, characterized by abnormalgrowth of cells (e.g., cancer). Other applications for compounds andconjugates of this invention include, but are not limited to, treatingosteoporosis, depression, anxiety, stress, phobias, panic, dysphoria,psychiatric disorders, and pain or as antiepileptics, antibacterials,diuretics and hypotensives, hypolipidemics, and anti-depressants.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-10 show the schemes for the synthesis of indolinobenzodiazepineand oxazolidinobenzodiazepine monomers, the representative linkers andthe dimers in the present invention.

FIG. 11 shows the scheme for the synthesis of the representative B-ringmodified indolinobenzodiazepine monomer.

FIG. 12 shows the scheme for the synthesis of the representativeisoindolinobenzodiazepine monomer.

FIG. 13 shows the scheme for the synthesis of the representative dimerwith the linker directly attached on the indolinobenzodiazepine moietyin the present invention.

FIGS. 14 and 15 show the schemes for the synthesis of the representativedimers containing (PEG)_(n) moieties on the linkers.

FIG. 16 shows the schemes for the synthesis of the representative mixedimine-amine and imine-amide indolinobenzodiazepine dimers.

FIG. 17 shows the scheme for the synthesis of the representativeIBD-poly(N-methylpyrrole-imidazole) conjugates.

FIGS. 18-19 show the synthetic scheme for the preparation of polypyrroloand polypyrrolo-imidazolo derivatives of the monomers.

FIG. 20 shows a scheme for the synthesis of piperidinylbenzodiazepinesbearing a hydrazone linker.

FIGS. 21-26 show the dose dependent in vitro antiproliferative activityof muB38.1-IGN-03, huN901-IGN-03, huN901-IGN-07, and muB38.1-IGN-10conjugates on antigen positive and antigen negative cancer cell lines.

FIG. 27 shows in vivo efficacy of huN901-IGN-07 conjugate in micebearing Molp-8 tumors.

FIGS. 28-30 show data that demonstrate that IGN-01, IGN-02, and IGN-09bind and covalently adduct to double stranded DNA containing guanineresidues on opposite strands.

FIG. 31 contains TABLE 1, which shows the IC₅₀ values for in vitroantiproliferative activity of indolinobenzodiazepine dimers andoxazolidinobenzodiazepine dimer on several cancer cell lines.

FIG. 32 contains TABLE 2, which shows the comparison of the IC₅₀ valuesfor in vitro antiproliferative activity of indolinobenzodiazepine dimerswith and without linkers.

FIGS. 33-36, 39, 42, 43, 44, 48, 49 and 50 show synthetic schemes forthe preparation of compounds of the present invention.

FIGS. 37, 38, 40 and 41, 45, 46, and 47 show synthetic schemes for thepreparation of linkable compounds of the present invention.

FIG. 51 shows the in vitro cytotoxicity of compounds of the presentinvention.

FIGS. 52, 54, 56, 57 and 58 show the in vitro cytotoxicity andspecificity of chB38.1 conjugates.

FIGS. 53 and 55 show the in vitro cytotoxicity and specificity ofhuMy9-6 conjugates.

FIG. 59 shows the in vivo anti-tumor activity of chB38.1 conjugate

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

DEFINITIONS

“Linear or branched alkyl” as used herein refers to a saturated linearor branched-chain monovalent hydrocarbon radical of one to twenty carbonatoms. Examples of alkyl include, but are not limited to, methyl, ethyl,1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, —CH₂CH(CH₃)₂), 2-butyl,2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl 4-methyl-2-pentyl3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like.

“Linear or branched alkenyl” refers to linear or branched-chainmonovalent hydrocarbon radical of two to twenty carbon atoms with atleast one site of unsaturation, i.e., a carbon-carbon, double bond,wherein the alkenyl radical includes radicals having “cis” and “trans”orientations, or alternatively, “E” and “Z” orientations. Examplesinclude, but are not limited to, ethylenyl or vinyl (—CH═CH₂), allyl(—CH₂CH═CH₂), and the like.

“Linear or branched alkynyl” refers to a linear or branched monovalenthydrocarbon radical of two to twenty carbon atoms with at least one siteof unsaturation, i.e., a carbon-carbon, triple bond. Examples include,but are not limited to, ethynyl, propynyl, 1-butyryl, 2-butyryl,1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like.

The terms “cyclic alkyl”, “cyclic alkenyl”, “cyclic alkynyl”,“carbocycle”, “carbocyclyl”, “carbocyclic ring” and “cycloalkyl” referto a monovalent non-aromatic, saturated or partially unsaturated ringhaving 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atomsas a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms can bearranged, for example, as a bicyclo[4,5], [5,5], [5,6] or [6,6] system,and bicyclic carbocycles having 9 or 10 ring atoms can be arranged as abicyclo[5,6] or [6,6] system, or as bridged systems such asbicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.Examples of monocyclic carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and thelike.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-18 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, indenyl, indanyl,1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 18 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus, and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, and azabicyclo[2.2.2]hexanyl. Spiromoieties are also included within the scope of this definition. Examplesof a heterocyclic group wherein ring atoms are substituted with oxo (═O)moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.

The term “heteroaryl” refers to a monovalent aromatic radical of 5- or6-membered rings, and includes fused ring systems (at least one of whichis aromatic) of 5-18 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl.

The heterocycle or heteroaryl groups may be carbon (carbon-linked) ornitrogen (nitrogen-linked) attached where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or O-carboline.

The heteroatoms present in heteroaryl or heterocyclcyl include theoxidized forms such as NO, SO, and SO₂.

The term “halo” or “halogen” refers to F, Cl, Br or I.

The term “compound” or “cytotoxic compound” or “cytotoxic agent” as usedherein is intended to include compounds for which a structure or formulaor any derivative thereof has been disclosed in the present invention ora structure or formula or any derivative thereof that has beenincorporated by reference. The term also includes, stereoisomers,geometric isomers, tautomers, solvates, metabolites, salts (e.g.,pharmaceutically acceptable salts) and prodrugs, and prodrug salts of acompound of all the formulae disclosed in the present invention. Theterm also includes any solvates, hydrates, and polymorphs of any of theforegoing. The specific recitation of “stereoisomers”, “geometricisomers”, “tautomers”, “solvates”, “metabolites”, “salt” “prodrug,”“prodrug salt,” “conjugates,” “conjugates salt,” “solvate,” “hydrate,”or “polymorph” in certain aspects of the invention described in thisapplication shall not be interpreted as an intended omission of theseforms in other aspects of the invention where the term “compound” isused without recitation of these other forms.

The term “conjugate” as used herein refers to a compound or a derivativethereof that is linked to a cell binding agent and is defined by ageneric formula: C-L-CBA, wherein C=compound, L=linker, and CBA=cellbinding agent.

The term “linkable to a cell binding agent” as used herein refers to thenovel benzodiazepine compounds (e.g., indolinobenzodiazepine oroxazolidinobenzodiazepine), derivates thereof or dimers thereofcomprising at least one linking group or a precursor thereof suitable tobond these compounds, derivatives thereof or dimers thereof to a cellbinding agent.

The term “precursor” of a given group refers to any group which may leadto that group by any deprotection, a chemical modification, or acoupling reaction.

The term “linked to a cell binding agent” refers to a conjugate moleculecomprising at least one the novel benzodiazepine compounds (e.g.,indolinobenzodiazepine or oxazolidinobenzodiazepine), derivates thereofor dimers thereof bound to a cell binding agent via a suitable linkinggroup or a precursor thereof.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomer” refers to compounds which have identicalchemical constitution and connectivity, but different orientations oftheir atoms in space that cannot be interconverted by rotation aboutsingle bonds.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as crystallization, electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

A substituent is “substitutable” if it comprises at least one carbon,sulfur, oxygen or nitrogen atom that is bonded to one or more hydrogenatoms. Thus, for example, hydrogen, halogen, and cyano do not fallwithin this definition.

If a substituent is described as being “substituted,” a non-hydrogensubstituent is in the place of a hydrogen substituent on a carbon,oxygen, sulfur or nitrogen of the substituent. Thus, for example, asubstituted alkyl substituent is an alkyl substituent wherein at leastone non-hydrogen substituent is in the place of a hydrogen substituenton the alkyl substituent. To illustrate, monofluoroalkyl is alkylsubstituted with a fluoro substituent, and difluoroalkyl is alkylsubstituted with two fluoro substituents. It should be recognized thatif there is more than one substitution on a substituent, eachnon-hydrogen substituent may be identical or different (unless otherwisestated).

If a substituent is described as being “optionally substituted,” thesubstituent may be either (1) not substituted, or (2) substituted. If acarbon of a substituent is described as being optionally substitutedwith one or more of a list of substituents, one or more of the hydrogenson the carbon (to the extent there are any) may separately and/ortogether be replaced with an independently selected optionalsubstituent. If a nitrogen of a substituent is described as beingoptionally substituted with one or more of a list of substituents, oneor more of the hydrogens on the nitrogen (to the extent there are any)may each be replaced with an independently selected optionalsubstituent. One exemplary substituent may be depicted as —NR′R,″wherein R′ and R″ together with the nitrogen atom to which they areattached, may form a heterocyclic ring. The heterocyclic ring formedfrom R′ and R″ together with the nitrogen atom to which they areattached may be partially or fully saturated. In one embodiment, theheterocyclic ring consists of 3 to 7 atoms. In another embodiment, theheterocyclic ring is selected from the group consisting of pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl andthiazolyl.

This specification uses the terms “substituent,” “radical,” and “group”interchangeably.

If a group of substituents are collectively described as beingoptionally substituted by one or more of a list of substituents, thegroup may include: (1) unsubstitutable substituents, (2) substitutablesubstituents that are not substituted by the optional substituents,and/or (3) substitutable substituents that are substituted by one ormore of the optional substituents.

If a substituent is described as being optionally substituted with up toa particular number of non-hydrogen substituents, that substituent maybe either (1) not substituted; or (2) substituted by up to thatparticular number of non-hydrogen substituents or by up to the maximumnumber of substitutable positions on the substituent, whichever is less.Thus, for example, if a substituent is described as a heteroaryloptionally substituted with up to 3 non-hydrogen substituents, then anyheteroaryl with less than 3 substitutable positions would be optionallysubstituted by up to only as many non-hydrogen substituents as theheteroaryl has substitutable positions. Such substituents, innon-limiting examples, can be selected from a linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, halogen,guanidinium [—NH(C═NH)NH₂], OR₇, NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxiderepresented by SOR′, a sulfone represented by —SO₂R′, a sulfite —SO₃, abisulfite —OSO₃, a sulfonamide represented by SO₂NRR′, cyano, an azido,—COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂ wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ areeach independently selected from H, linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, arylhaving from 6 to 10 carbon atoms, heterocyclic ring having from 3 to 10carbon atoms.

The term “prodrug” as used in this application refers to a precursor orderivative form of a compound of the invention that is capable of beingenzymatically or hydrolytically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast(1986) and Stella et al., “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). The prodrugs of this invention include,but are not limited to, ester-containing prodrugs, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containingprodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, .beta.-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs, optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, compounds of the invention and chemotherapeutic agents suchas described above.

The term “prodrug” is also meant to include a derivative of a compoundthat can hydrolyze, oxidize, or otherwise react under biologicalconditions (in vitro or in vivo) to provide a compound of thisinvention. Prodrugs may only become active upon such reaction underbiological conditions, or they may have activity in their unreactedforms. Examples of prodrugs contemplated in this invention include, butare not limited to, analogs or derivatives of compounds of any one ofthe formulae disclosed herein that comprise biohydrolyzable moietiessuch as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. Other examples of prodrugs includederivatives of compounds of any one of the formulae disclosed hereinthat comprise —NO, —NO₂, —ONO, or —ONO₂ moieties. Prodrugs can typicallybe prepared using well-known methods, such as those described byBurger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982(Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, ThePharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed.1992, “Biotransformation of Drugs”.

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide”, “biohydrolyzable ester”, “biohydrolyzablecarbamate”, “biohydrolyzable carbonate”, “biohydrolyzable ureide” and“biohydrolyzable phosphate analogue” mean an amide, ester, carbamate,carbonate, ureide, or phosphate analogue, respectively, that either: 1)does not destroy the biological activity of the compound and confersupon that compound advantageous properties in vivo, such as uptake,duration of action, or onset of action; or 2) is itself biologicallyinactive but is converted in vivo to a biologically active compound.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable estersinclude, but are not limited to, lower alkyl esters, alkoxyacyloxyesters, alkyl acylamino alkyl esters, and choline esters. Examples ofbiohydrolyzable carbamates include, but are not limited to, loweralkylamines, substituted ethylenediamines, amino acids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines. Particularly favored prodrugs and prodrug salts arethose that increase the bioavailability of the compounds of thisinvention when such compounds are administered to a mammal.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

As used herein, the term “solvate” means a compound which furtherincludes a stoichiometric or non-stoichiometric amount of solvent suchas water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate,acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like,bound by non-covalent intermolecular forces. Solvates or hydrates of thecompounds are readily prepared by addition of at least one molarequivalent of a hydroxylic solvent such as methanol, ethanol,1-propanol, 2-propanol or water to the compound to result in solvationor hydration of the imine moiety.

The terms “abnormal cell growth” and “proliferative disorder” are usedinterchangeably in this application. “Abnormal cell growth”, as usedherein, unless otherwise indicated, refers to cell growth that isindependent of normal regulatory mechanisms (e.g., loss of contactinhibition). This includes, for example, the abnormal growth of: (1)tumor cells (tumors) that proliferate by expressing a mutated tyrosinekinase or overexpression of a receptor tyrosine kinase; (2) benign andmalignant cells of other proliferative diseases in which aberranttyrosine kinase activation occurs; (3) any tumors that proliferate byreceptor tyrosine kinases; (4) any tumors that proliferate by aberrantserine/threonine kinase activation; and (5) benign and malignant cellsof other proliferative diseases in which aberrant serine/threoninekinase activation occurs.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, acute leukemia, as well as head/brain andneck cancer.

A “therapeutic agent” encompasses both a biological agent such as anantibody, a peptide, a protein, an enzyme or a chemotherapeutic agent. A“chemotherapeutic agent” is a chemical compound useful in the treatmentof cancer. Examples of chemotherapeutic agents include Erlotinib(TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, MillenniumPharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer),Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis),PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU(5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth),Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafarnib (SCH66336), Sorafenib (BAY43-9006, Bayer Labs), and Gefitinib (IRESSA®,AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such asthiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammal1 and calicheamicinomegal1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France);chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate;daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®.; PROLEUKIN® rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and (x) pharmaceutically acceptablesalts, acids and derivatives of any of the above. Other anti-angiogenicagents include MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9(matrix-metalloproteinase 9) inhibitors, COX-II (cyclooxygenase II)inhibitors, and VEGF receptor tyrosine kinase inhibitors. Examples ofsuch useful matrix metalloproteinase inhibitors that can be used incombination with the present compounds/compositions are described in WO96/33172, WO 96/27583, EP 818442, EP 1004578, WO 98/07697, WO 98/03516,WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, EP 606,046, EP931,788, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO99/07675, EP 945864, U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510,and EP 780,386, all of which are incorporated herein in their entiretiesby reference. Examples of VEGF receptor tyrosine kinase inhibitorsinclude4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)qu-inazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-1-quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), and compounds such asthose disclosed in PCT Publication Nos. WO 97/22596, WO 97/30035, WO97/32856, and WO 98/13354).

Other examples of chemotherapeutic agents that can be used incombination with the present compounds include inhibitors of PI3K(phosphoinositide-3 kinase), such as those reported in Yaguchi et al(2006) Jour. of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No.7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat.No. 6,608,053; U.S. Pat. No. 6,838,457; U.S. Pat. No. 6,770,641; U.S.Pat. No. 6,653,320; U.S. Pat. No. 6,403,588; WO 2006/046031; WO2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070; U.S. Pat. No. 6,703,414; and WO 97/15658, all of which areincorporated herein in their entireties by reference. Specific examplesof such PI3K inhibitors include SF-1126 (PI3K inhibitor, SemaforePharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3Kinhibitor, Exelixis, Inc.).

A “metabolite” is a product produced through metabolism in the body of aspecified compound, a derivative thereof, or a conjugate thereof, orsalt thereof. Metabolites of a compound, a derivative thereof, or aconjugate thereof, may be identified using routine techniques known inthe art and their activities determined using tests such as thosedescribed herein. Such products may result for example from theoxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation,esterification, deesterification, enzymatic cleavage, and the like, ofthe administered compound. Accordingly, the invention includesmetabolites of compounds, a derivative thereof, or a conjugate thereof,of the invention, including compounds, a derivative thereof, or aconjugate thereof, produced by a process comprising contacting acompound, a derivative thereof, or a conjugate thereof, of thisinvention with a mammal for a period of time sufficient to yield ametabolic product thereof.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The term “protecting group” or “protecting moiety” refers to asubstituent that is commonly employed to block or protect a particularfunctionality while reacting other functional groups on the compound, aderivative thereof, or a conjugate thereof. For example, an“amino-protecting group” or an “amino-protecting moiety” is asubstituent attached to an amino group that blocks or protects the aminofunctionality in the compound. Suitable amino-protecting groups includeacetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a“hydroxy-protecting group” refers to a substituent of a hydroxy groupthat blocks or protects the hydroxy functionality. Suitable protectinggroups include acetyl and silyl. A “carboxy-protecting group” refers toa substituent of the carboxy group that blocks or protects the carboxyfunctionality. Common carboxy-protecting groups includephenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl,2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl,2-(p-nitrophenylsulfenyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyland the like. Common thiol-protecting groups include those that convertthe thiol into a thioester, such as acetyl, benzoyl or trifluoroacetyl,into a thioether, such as benzyl, t-butyl, triphenylmethyl,9-fluorenylmethyl, methoxymethyl, 2-tetrahydropyranyl or silyl, into adisulfide, such as methyl, benzyl, t-butyl, pyridyl, nitropyridyl,phenyl, nitrophenyl or dinitrophenyl, into a thiocarbonate, such ast-butoxycarbonyl, into a thiocarbamate, such as N-ethyl. For a generaldescription of protecting groups and their use, see P. G. M. Wuts & T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,New York, 2007.

For novel benzodiazepines of formula (I) and (II),

wherein:the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H, or an amine protecting moiety that converts the compoundinto a prodrug that can be transformed into the free amine in vitro orin vivo;Y is selected from —OR, an ester represented by —OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxylamine represented by NR′R″, amide represented by —NRCOR′, apeptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand are selected from H, substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbon atoms apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000, a 5- or 6-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, a5 to 18 membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, a 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P wherein the substituent is selected from halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selectedfrom H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from1 to 10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), whereinn is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur. a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to6 heteroatoms selected from O, S, N and P and R₁₀ optionally is SR₁₃ orCOR₁₃, wherein R₁₃ is selected from linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, a 5-or 6-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, a 5 to 18membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, 3 to 18-membered heterocyclic ring having1 to 6 heteroatoms selected from O, S, N and P and R₁₁ can also be OR₁₄,wherein R₁₄ is H or has the same definition as R, optionally, R″ is anOH;W is C═O, C═S, CH₂, BH (B=Boron), SO or SO₂;R₁, R₂, R₃, R₄, are each independently selected from H, substituted orunsubstituted linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, or a substituentselected from a halogen, OR₇, NR₈R₉, NO₂, NRCOR′, SR₁0, a sulfoxiderepresented by SOR′, a sulfone represented by —SO₂R′, a sulfite —SO₃, abisulfite —OSO₃, a sulfonamide represented by SO₂NRR′, cyano, an azido,guanidinium [—NH(C═NH)NH₂], —COR₁₁, —OCOR₁₁ or —OCONR₁₁R₁₂ wherein R₇,R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined above, optionally, any one ofR₁, R₂, R₃, R₄ is a linking group that enables linkage to a cell bindingagent via a covalent bond or is selected from a polypyrrolo,poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl,poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing alinking group that enables linkage to a cell binding agent;R₅ is selected from OR₁₅, CRR′OH, SH, CRR′SH, NHR₁₅ or CRR′NHR₁₅,wherein R₁₅ has the same definition as R., R and R′ have the samedefinition as given above; optionally, R₅ is a linking group thatenables linkage to a cell binding agent via a covalent bond or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent;R₆ is OR, SR, NRR′, wherein R and R′ have the same definition as givenabove, or optionally R₆ is a linking group;Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000; or their pharmaceutically acceptable solvates, salts, hydratesor hydrated salts, their optical isomers, racemates, diastereomers,enantiomers of these compounds.provided that the compound has no more than one linking group thatenables linkage to a cell binding agent via a covalent bond.

In one preferred embodiment, the double line

between N and C represents a double bond and X is absent and Y is H, orthe double line

between N and C represents a single bond wherein X is H and Y isselected from —OR, a sulfite —SO₃, or an amine protecting moiety thatconverts the compound into a prodrug;

W is C═O, CH₂, or SO₂;

R₁, R₂, R₃, R₄, are each H; optionally, independently, any one of R₁,R₂, R₃ and R₄ can be a linking group that enables linkage to a cellbinding agent via a covalent bond;

R₅ is selected from OR₁₅, CRR′OH, SH, CRR′SH, NHR₁₅ or CRR′NHR₁₅,wherein R₁₅ is H or has the same definition as given above for R, or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent, R and R′ have the same definition as given above;R₆ is OCH₃;Z is selected from (CH₂)_(n), wherein n is 1 or 2, NH, NCH₃ or S; ortheir pharmaceutically acceptable solvates, salts, hydrates or hydratedsalts, their optical isomers, racemates, diastereomers, enantiomers orthe polymorphic crystalline structures of these compounds.

In a preferred embodiment, compounds of formula (I) and (II) arecompounds of formulae (VII), (VIII) or (IX):

wherein the substituents are described as above; or theirpharmaceutically acceptable solvates, salts, hydrates or hydrated salts,their optical isomers, racemates, diastereomers, enantiomers or thepolymorphic crystalline structures of these compounds.

For the novel benzodiazepines of formula (III), in which the diazepinering (B) is fused with a heterocyclic ring (C), wherein the heterocyclicring is monocyclic,

wherein:the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting moiety that converts the compoundinto a prodrug;Y is selected from —OR, an ester represented by —OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxylamine represented by NR′R″, amide represented by —NRCOR′, apeptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand selected from H, substituted or unsubstituted linear, branched orcyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000, a 5- or 6-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, a5 to 18 membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfurheteroaryl comprising of 5- or 6-memberedrings, including fused ring systems (at least one of which is aromatic)of 5-18 atoms, containing one or more heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selectedfrom O, S, N and P, wherein the substituent is selected from halogen,OR₇, NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, asulfone represented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, asulfonamide represented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ orOCONR₁₁R₁₂, wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independentlyselected from H, linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, a 5- or6-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, a 5 to 18membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfurheteroaryl comprising of 5- or 6-memberedrings, including fused ring systems (at least one of which is aromatic)of 5-18 atoms, containing one or more heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selectedfrom O, S, N and P and R₁₀ optionally is SR₁₃ or COR₁₃, wherein R₁₃ isselected from linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, a 5- or6-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, a 5 to 18membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfurheteroaryl comprising of 5- or 6-memberedrings, including fused ring systems (at least one of which is aromatic)of 5-18 atoms, containing one or more heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbonatoms, 3 to 18-membered heterocyclic ring having 1 to 6 heteroatomsselected from O, S, N and P and R₁₁ can also be OR₁₄, wherein R₁₄ is Hor has the same definition as R, optionally R″ is OH;W is C═O, C═S, CH₂, BH, SO or SO₂;R₅ is selected from OR₁₅, CRR′OH, SH, CRR′SH, NHR₁₅ or CRR′NHR₁₅,wherein R₁₅ is H or has the same definition as R. or is a linking groupthat enables linkage to a cell binding agent via a covalent bond or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent; is selected from a poly-pyrrolo, poly-indolyl,poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl orpolyimidazolo-indolyl unit optionally bearing a linking group thatenables linkage to a cell binding agent, optionally, R₅ is a linkinggroup that enables linkage to a cell binding agent via a covalent bond;R₆ is OR, SR or NRR′, wherein R and R′ have the same definition as givenabove, optionally R₆ is a linking group;X′ is CH₂, NR, CO, BH, SO or SO₂;Y′ is O, CH₂, NR or S;Z′ is CH₂ or (CH₂)_(n), wherein n is 2, 3 or 4; or theirpharmaceutically acceptable solvates, salts, hydrates or hydrated salts,their optical isomers, racemates, diastereomers, enantiomers or thepolymorphic crystalline structures of these compounds;provided that the compound has no more than one linking group thatenables linkage to a cell binding agent via a covalent bond.

In one preferred embodiment, the double line

between N and C represents a double bond and X is absent and Y═H, or thedouble line

between N and C represents a single bond wherein X is H and Y isselected from —OR, a sulfite —SO₃, or an amine protecting moiety thatconverts the compound into a prodrug;

W is C═O, CH₂, or SO₂;

R₅ is selected from OR₁₅, CRR′OH, SH, CRR′SH, NHR₁5 or CRR′NHR₁₅,wherein R₁₅ is H or has the same definition as given above for R, or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrolloindolyl or polyimidazoloindolyl unitoptionally bearing a linking group that enables linkage to a cellbinding agent;R₆ is OCH₃;X′ is selected from CH₂, or C═O;Y′ is O, CH₂, NR or S;Z′ is (CH₂)_(n), wherein n is 1 or 2, provided that X′, Y′ and Z′ arenot all CH₂ at the same time; or their pharmaceutically acceptablesolvates, salts, hydrates or hydrated salts, their optical isomers,racemates, diastereomers, enantiomers or the polymorphic crystallinestructures of these compounds.

In a preferred embodiment, compound of formula III is represented by acompound of formula (X) or (XI),

wherein the substituents are described as above; or theirpharmaceutically acceptable solvates, salts, hydrates or hydrated salts,their optical isomers, racemates, diastereomers, enantiomers or thepolymorphic crystalline structures of these compounds.

For the cytotoxic dimers represented by formulas (IV), (V) and (VI)

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting moiety that converts the compoundinto a prodrug;Y is selected from —OR, an ester represented by —OCOR′, a carbonaterepresented by —OCOOR′, a carbamate represented by —OCONR′R″, an amineor a hydroxylamine represented by NR′R″, amide represented by —NRCOR′, apeptide represented by NRCOP, wherein P is an amino acid or apolypeptide containing between 2 to 20 amino acid units, a thioetherrepresented by SR′, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a halogen,cyano, an azido, or a thiol, wherein R, R′ and R″ are same or differentand are selected from H, substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to2000, a 5- or 6-membered heteroaryl ring containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, a5 to 18 membered fused ring system, wherein at least one of the rings isaromatic, containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P wherein the substituent is selected from halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selectedfrom H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from1 to 10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), whereinn is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms, 3 to 10-membered heterocyclic ring having 3to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected fromO, S, N and P and R₁₀ is optionally SR₁₃ or COR₁₃, wherein R₁₃ isselected from linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a 5- or 6-membered heteroaryl ringcontaining one or more heteroatoms independently selected from nitrogen,oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein atleast one of the rings is aromatic, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, aryl havingfrom 6 to 18 carbon atoms, 3 to 18-membered heterocyclic ring having 1to 6 heteroatoms selected from O, S, N and P, optionally R₁₁ is OR₁₄,wherein R₁₄ has the same definition as R, optionally R″ is OH;W is C═O, C═S, CH₂, BH, SO or SO₂;R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently selectedfrom H, substituted or unsubstituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit (—OCH₂CH₂)n, wherein n is an integer from 1 to 2000, or asubstituent selected from a halogen, guanidinium [—NH(C═NH)NH₂], OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂wherein R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined above, optionally,any one of R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, or R₄′ is a linking group thatenables linkage to a cell binding agent via a covalent bond or isselected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally bearing a linking group that enables linkage to a cellbinding agent,Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;R₆ is OR, SR or NRR′, wherein R and R′ have the same definition as givenabove, optionally R₆ is a linking group;X′ is selected from CH₂, NR, CO, BH, SO or SO₂ wherein R has the samedefinition as given above;Y′ is O, CH₂, NR or S, wherein R has the same definition as given above;Z′ is CH₂ or (CH₂)_(n), wherein n is 2, 3 or 4, provided that X′, Y′ andZ′ are not all CH₂ at the same time;A and A′ are the same or different and are selected from O, —CRR′O, S,—CRR′S, —NR₁₅ or CRR′NHR₁₅, wherein R and R′ have the same definition asgiven above and wherein R₁₅ has the same definition as R.D and D′ are same or different and independently selected from linear,branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms, optionally substituted with any one of halogen, OR₇, NR₈R₉, NO₂,NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfone represented by—SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamide represented bySO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂, wherein thedefinitions of R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined above, or apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;L is an optional phenyl group or 3 to 18-membered heterocyclic ringhaving 1 to 6 heteroatoms selected from O, S, N and P that is optionallysubstituted, wherein the substituent is a linking group that enableslinkage to a cell binding agent via a covalent bond, or is selected fromlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms, optionally substituted with any one of halogen, OR₇,NR₈R₉, NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein the definitions of R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are as definedabove, a polyethylene glycol unit (—OCH₂CH₂)n, wherein n is an integerfrom 1 to 2000; optionally, L itself is a linking group that enableslinkage to a cell binding agent via a covalent bond; or theirpharmaceutically acceptable solvates, salts, hydrates or hydrated salts,their optical isomers, racemates, diastereomers, enantiomers or thepolymorphic crystalline structures of these compounds; provided that thecompound has no more than one linking group that enables linkage to acell binding agent via a covalent bond.

In one preferred embodiment, the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting group that converts the compoundinto a prodrug;

Y is selected from —OR, NR′R″, a sulfite —SO₃, or a bisulfite —OSO₃,wherein R is selected from H, linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, aryl having from6 to 10 carbon atoms, heterocyclic ring having from 3 to 10 carbonatoms;W is C═O, CH₂ or SO₂;R₁, R₂, R₃, R₄, R₁′. R₂′. R₃′ and R₄′ are each independently selectedfrom H, NO₂ or a linking group that enables linkage to a cell bindingagent via a covalent bond;R₆ is OR₁₈, wherein R₁₈ has the same definition as R;Z is selected from (CH₂)_(n), wherein n is 1, 2 or 3, CR₁₅R₁₆, NR₁₇, Oor S, wherein R₁₅, R₁₆ and R₁₇ are each independently selected from H,linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000;X′ is selected from CH₂, or C═O;Y′ is O, NR, or S, wherein R is defined as above;Z′ is CH₂ or (CH₂)₂;A and A′ are each O;D and D′ are same or different and independently selected from linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms;L is an optional phenyl group or a heterocycle ring having from 3 to 10carbon atoms that is optionally substituted, wherein the substituent isa linking group that enables linkage to a cell binding agent via acovalent bond, or is selected from linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, optionallysubstituted with any one of halogen, OR₇, NR₈R₉, NO₂, NRCOR′, SR₁₀, asulfoxide represented by SOR′, a sulfone represented by —SO₂R′, asulfite —SO₃, a bisulfite —OSO₃, a sulfonamide represented by SO₂NRR′,cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂, a polyethylene glycolunit (—OCH₂CH₂)n, wherein n is an integer from 1 to 2000; optionally, Litself is a linking group that enables linkage to a cell binding agentvia a covalent bond; or their pharmaceutically acceptable solvates,salts, hydrates or hydrated salts, their optical isomers, racemates,diastereomers, enantiomers or the polymorphic crystalline structures ofthese compounds.

In another preferred embodiment, the compound of formula (IV), (V) or(VI) is represented by compounds of formulae (XII) and (XIII):

wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting group that converts the compoundinto a prodrug; Y is selected from OH, an ether represented by —OR,NR′R″, a sulfite —SO₃, or a bisulfite —OSO₃, wherein R, R′ and R″ areselected from linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms;one of R₂, R₃, R₂′ and R₃′ is a linking group that enables linkage to acell binding agent via a covalent bond and the others are H, NRCOR′ orNO₂;R₆ is OR, wherein R has the same definition as above;Z is CH₂ or NR, wherein R has the same definition as above;A is O or NR₁₅;L is (CH₂)_(nn), wherein nn is 0 or an integer between 1 and 5, or asubstituted or unsubstituted alkyl or alkenyl having from 2 to 4 carbonatoms, wherein the substituent is selected from halogen, OR₇, NR₈R₉,NO₂, NRCOR′, SR₁₀, a sulfoxide represented by SOR′, a sulfonerepresented by —SO₂R′, a sulfite —SO₃, a bisulfite —OSO₃, a sulfonamiderepresented by SO₂NRR′, cyano, an azido, —COR₁₁, OCOR₁₁ or OCONR₁₁R₁₂,wherein R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₅ has the same definition asgiven above, optionally, L itself is a linking group that enableslinkage to a cell binding agent via a covalent bond;one of L′, L″ or L′″ is a linking group that enables linkage to a cellbinding agent, while the others are H; preferably L′ is the linkinggroup; andG is CH or N or their pharmaceutically acceptable solvates, salts,hydrates or hydrated salts, their optical isomers, racemates,diastereomers, enantiomers or the polymorphic crystalline structures ofthese compounds.

In yet another preferred embodiment, the compound of formula (IV), (V)or (VI) is represented by compounds of formulae from formulae (XIV) and(XV):

wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is H, and when it is a singlebond, X is H or an amine protecting group that converts the compoundinto a prodrug; Y is selected from OH, an ether represented by —OR, asulfite —SO₃, or a bisulfite —OSO₃, wherein R is selected from linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms;nn is 0 or an integer from 1 to 5;One of R₂, R₃, R₂′ and R₃′ is a linking group that enables linkage to acell binding agent via a covalent bond and the others are H, NRCOR′, orNO₂;one of L′, L″ or L′″ is a linking group that enables linkage to a cellbinding agent, provided that when one of L′, L″ or L′″ is a linkinggroup others are H (e.g., if L′ is a linker, then L″ and L′″ are H)G is CH or N or their pharmaceutically acceptable solvates, salts,hydrates or hydrated salts, their optical isomers, racemates,diastereomers, enantiomers or the polymorphic crystalline structures ofthese compounds.

In order to link the cytotoxic compounds (e.g., indolinobenzodiazepineor oxazolidinobenzodiazepine), derivatives thereof, or dimers thereof ofthe present invention to the cell-binding agent, the cytotoxic compoundcomprises a linking moiety. While a linker that connects two moieties isbifunctional, one end of the linker moiety can be first reacted with thecytotoxic compound to provide the compound bearing a monofunctionallinking group, which can then react with a cell binding agent.Alternatively, one end of the linker moiety can be first reacted withthe cell binding agent to provide the cell binding agent bearing amonofunctional linking group, which can then react with a cytotoxiccompound. The linking moiety contains a chemical bond that allows forthe release of the cytotoxic moiety at a particular site. Suitablechemical bonds are well known in the art and include disulfide bonds,thioether bonds, acid labile bonds, photolabile bonds, peptidase labilebonds and esterase labile bonds (see for example U.S. Pat. Nos.5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497;7,276,499; 7,368,565; 7,388,026 and 7,414,073). Preferred are disulfidebonds, thioether and peptidase labile bonds. Other linkers that can beused in the present invention include non-cleavable linkers, such asthose described in are described in detail in U.S. publication number20050169933, or charged linkers or hydrophilic linkers and are describedin provisional patent applications, 61/049,291, filed Apr. 30, 2008,61/147,966, filed Jan. 28, 2009, and 61/049,289, filed Apr. 30, 2008,each of which is expressly incorporated herein by reference.

The compounds of formula (I), (II), and (III) (i.e., monomers) can belinked through R₁, R₂, R₃, R₄ or R₅. Of these, preferred linkable groupsare R₂, R₃, and R₅, and the most preferred linkable group is R₅.Examples of suitable substituents at R₁, R₂, R₃, R₄ and R₅ for compoundsof formula (I), (II) and (III) include, but are not limited to:

—OH,

—O(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(indolo)_(p′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(indolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(indolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—SH,

—S(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(indolo)_(p′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(indolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)A″_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NH₂,

—NR₂₈(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(indolo)_(p′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(imidazole)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″—(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(indolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″—(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(indolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″—(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₂₈(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(indolo)_(p′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(indolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

(CR₂₀R₂₁)_(m)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

(CR₂₀R₂₁)_(m)(pyrrolo)_(q′)(imidazolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

(CR₂₀R₂₁)_(m)(imidazolo)_(q′)(indolo)_(q″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

wherein:

m, n, p, q, m′, n′, p′, q′, q″, are integer from 1 to 10 and can be 0;

t, m″, n″ and p″ are 0 or 1;

X″ is selected from OR₃₆, SR₃₇, NR₃₈R₃₉, wherein R₃₆, R₃₇, R₃₈, R₃₉ areH, or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1to 20 carbon atoms and, or, a polyethylene glycol unit —(OCH₂CH₂)_(n),optionally R₃₇ is a thiol protecting group, orwhen t=1, COX″ forms a reactive ester selected from N-hydroxysuccinimideesters, N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters,para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl estersand their derivatives, wherein said derivatives facilitate amideformation;Y″ is absent or is selected from O, S, S—S or NR₃₂, wherein R₃₂ has thesame definition as given above for R, orwhen Y″ is not S—S and t=0, X″ is selected from a maleimido group, ahaloacetyl group or SR₃₇, wherein R₃₇ has the same definition as above;A″ is an amino acid selected from glycine, alanine, leucine, valine,lysine, citrulline and glutamate or a polypeptide containing between 2to 20 amino acid units;R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇ are the same or different and areH or a linear or branched alkyl having from 1 to 5 carbon atoms;R₂₈ is H or alkyl;R₂₉ and R₃₀ are the same or different and are H or alkyl from 1 to 5carbon atoms;optionally, one of R₄₀ and R₄₁ is a negatively or positively chargedfunctional group and the other is H or alkyl, alkenyl, alkynyl having 1to 4 carbon atoms.

The compounds of formula (IV), (V), (VI), (VII), (XII) and (XIII) (i.e.,dimers) can be linked through R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, L′,L″, L′″. Of these, preferred linkable groups are R₂′, R₃′, R₄′, L′, L″,L′″ and most preferred linkable groups are R₂′, R₃′ and L′. Examples oflinking groups for compounds of formula (IV), (V), (VI), (VII), (XII)and (XIII) include, but are not limited to:

—O(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—O(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(m′)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—S(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉N—NR₃₀)_(n″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(CR₂₆═CR₂₇)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

wherein:

m, n, p, q, m′, n′, t′ are integer from 1 to 10, or are optionally 0;

t, m″, n″ and p″ are 0 or 1;

X″ is selected from OR₃₆, SR₃₇, NR₃₈R₃₉, wherein R₃₆, R₃₇, R₃₈, R₃₉ areH, or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1to 20 carbon atoms and, or, a polyethylene glycol unit —(OCH₂CH₂)_(n),R₃₇, optionally, is a thiol protecting groupwhen t=1, COX″ forms a reactive ester selected from N-hydroxysuccinimideesters, N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters,para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl estersand their derivatives, wherein said derivatives facilitate amide bondformation;Y″ is absent or is selected from O, S, S—S or NR₃₂, wherein R₃₂ has thesame definition as given above for R, orwhen Y″ is not S—S and t=0, X″ is selected from a maleimido group, ahaloacetyl group or SR₃₇, wherein R₃₇ has the same definition as above;A″ is an amino acid selected from glycine, alanine, leucine, valine,lysine, citrulline and glutamate or a polypeptide containing between 2to 20 amino acid units;R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, and R₂₇ are the same or different andare H or a linear or branched alkyl having from 1 to 5 carbon atoms;R₂₉ and R₃₀ are the same or different and are H or alkyl from 1 to 5carbon atoms;R₃₃ is H or linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 12 carbon atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n), orR₃₃ is —COR₃₄, —CSR₃₄, —SOR₃₄, or —SO₂R₃₄, whereinR₃₄ is H or linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 20 carbon atoms or, a polyethylene glycol unit —(OCH₂CH₂)_(n);andone of R₄₀ and R₄₁ is optionally a negatively or positively chargedfunctional group and the other is H or alkyl, alkenyl, alkynyl having 1to 4 carbon atoms.

Further, while the synthesis of cytotoxic compounds (e.g.,indolinobenzodiazepine or oxazolidinobenzodiazepine), derivativesthereof, or dimers thereof bearing a linking moiety is described belowin terms of an amide, thioether or disulfide bond containing linkingmoieties at the L′ (in the compound of formula XIII) or R₃ (in thecompound of formula XII) positions, one of skill in the art willunderstand that linking moieties at other positions and with otherchemical bonds, as described above, can also be used with the presentinvention.

The structures of representative compounds, representative conjugatesand claimed compounds in the examples of the present invention are shownin Tables 3˜9:

TABLE 3 Structures of representative compounds of the present invention.

Note: Z″ = H, SMe, SPy, SPy—NO₂, Ac; X′′′ = NHS;

TABLE 4 Structures of representative compounds of the present invention(Continued).

Note: Z″ = H, SMe, SPy, SPy—NO₂, Ac; X′′′ = NHS;

TABLE 5 Structures of representative compounds of the present invention(Continued).

TABLE 6 Structures of representative conjugates of the presentinvention.

TABLE 7 Structures of compounds from the examples of the presentinvention. Structure Compound No. Example No.

6 1

7 1

8 1

12 2

13 2

14 2

15 3

18 4

19 5

34 6

TABLE 8 Structures of compounds from the examples of the presentinvention (Continued). Structure Compound No. Example No.

35 6

36 6

39 6

40 6

41 7

42 7

43 7

44 7

45 7

TABLE 9 Structures of compounds from the examples of the presentinvention (Continued). Structure Compound No. Example No.

46 7

48 8

49 8

51 9

125 10

126 10

127 10Synthesis of Cytotoxic Compounds

The process of preparation of a representative monomer compound of thepresent invention, exemplified by indolinobenzodiazepine compound 8, isshown in FIG. 1. Starting from commercially availableindoline-2-carboxylic acid 1, its methyl ester 2 was prepared inquantitative yield by reaction with thionyl chloride in methanol. Methylindoline-2-carboxylate 2 was coupled with the acid chloride 4, ordirectly with acid 3, to furnish the amide 5, which was further reducedwith diisobutylaluminum hydride (DIBAL) to the aldehyde 6. While, manymethods can be used to reduce the nitro functional group of formula 5 tothe corresponding amino group, in this example sodium dithionite wasused to conveniently convert to aldehyde 6 to the ring closed compound 7after further treatment with methanol under acidic conditions. Thebenzyl protecting group was removed to furnish monomer 8.

The process of preparation of the oxazolidinobenzodiazepine monomercompound of formula 14 of the invention is shown in FIG. 2. Startingfrom commercially available compound 9, its methyl ester 10 was preparedin quantitative yield by treatment with thionyl chloride in methanol.Compound 10 was deprotected followed by coupling with the acetylchloride 4 or directly with acid 3 to furnish the amide 11, which wasfurther converted to the aldehyde 12. Reduction of the nitro group wasaccomplished by treatment with sodium dithionite followed by efficientconversion to the ring closed compound 13 after further treatment withmethanol under acidic conditions. The benzyl protecting group wasremoved to furnish monomer 14.

The process of preparation of representative dimer compounds of thepresent invention is shown in FIGS. 3-5 and 7. The dimers were preparedby reacting of the monomers of formula 8 or formula 14 with compoundswhich possesses two leaving groups such as Br, I, triflate, mesylate ortosylate.

Dimers which possess linkers that can react with antibodies are preparedby converting the methyl esters to the corresponding reactive esters ofa leaving group such as, but not limited to, N-hydroxysuccinimideesters, N-hydroxyphtalimide esters, N-hydroxy sulfo-succinimide esters,para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters.Representative examples for the synthesis of the linkable dimers areshown in FIG. 8. Synthesis of dimers that bear a thiol or disulfidemoiety to enable linkage to cell binding agents via reducible ornon-reducible bonds is shown in FIGS. 9 and 10. The B ring modifiedmonomer 58 devoid of a carbonyl group is achieved from the benzylalcohol compound 52 by the steps shown in FIG. 11. The isoindolinomonomer 66 can be prepared from isoindole 59 as outlined in FIG. 12. Thelinker can also be attached directly to the indolino moiety. Methylindolino-2-carboxylate can be converted into the linkable dimer 82 viathe synthetic steps shown in FIG. 13. The synthesis of linkable dimersbearing a PEG moiety is shown in FIGS. 14 and 15.

Thus in one aspect, the invention provides a process for the preparationof the indolinobenzodiazepine (IBD) monomer of formula (I) (FIG. 1), theprocess comprising the steps of:

a) coupling compound of formula (1) and compound of formula (2) to givecompound of formula (3);

b) converting compound of formula (3) into aldehyde of formula (4); and

c) converting compound of formula (4) into compound of formula (I),

wherein LG is a leaving group; W′ is COOR or CH₂OW″, wherein R has thesame definition as above and W″ is a protecting group; R₁, R₂, R₃, R₄,R₅, R₆, W, Z, X, Y and

have the same definition as described above.

Another aspect of the invention provides a process for the preparationof compound of formula (II) comprising the steps of:

a) coupling compound of formula (1) and compound of formula (5) to givecompound of formula (6);

b) converting compound of formula (6) into aldehyde of formula (7); and

c) converting compound of formula (7) into compound of formula (II),

wherein LG is a leaving group; W′ is COOR or CH₂OW″, wherein R has thesame definition as above and W″ is a protecting group; R₁, R₂, R₃, R₄,R₅, R₆, W, X, Y and

have the same definition as above.

Another aspect of the invention provides a process for the preparationof compound of formula (III) comprising steps of:

a) coupling compound of formula (1) and compound of formula (8) to givecompound of formula (9);

b) converting compound of formula (9) into aldehyde of formula (10); and

c) converting compound of formula (10) into compound of formula (II),

wherein LG is a leaving group; W′ is COOR or CH₂OW″, wherein R has thesame definition as above and W″ is a protecting group; R₅, R₆, W, X, Y,X′, Y′, Z′ and

have the same definition as above.

Another aspect of the invention provides a process for the preparationof compound of formula (Another aspect of the invention provides aprocess for the preparation of compound of formula (IV) comprising thesteps of:

coupling compound of formula (11), compound of formula (11)′ andcompound of formula (12) to give compound of formula (IV),

wherein LG is a leaving group; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, R₆,W, X, Y, Z, A, A′, D, D′, L and

have the same definition as above.

Another aspect of the invention provides an alternative process for thepreparation of compound of formula (IV) of the present inventioncomprising steps of:

a) converting compound of formula (15) into aldehyde of formula (16);and

b) converting compound of formula (16) into compound of formula (IV),

wherein W′ is COOR or CH₂OW″, wherein R has the same definition as aboveand W″ is a protecting group; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, R₆, W,X, Y, Z, A, A′, D, D′, L and

have the same definition as above.Another aspect of the invention provides a process for the preparationof compound of formula (V) comprising the step of coupling compound offormula (13), compound of formula (13)′ and compound of formula (12) togive compound of formula (V),

wherein LG is a leaving group; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, R₆,W, X, Y, A, A′, D, D′, L and

have the same definition as above.

Another aspect of the invention provides an alternative process for thepreparation of compound of formula (V) of the invention comprising thesteps of:

a) converting compound of formula (17) into aldehyde of formula (18);and

b) converting compound of formula (18) into compound of formula (V),

wherein W′ is COOR or CH₂OW″, wherein R has the same definition as aboveand W″ is a protecting group; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, R₆, W,X, Y, A, A′, D, D′, L and

have the same definition as above.

Another aspect of the invention provides a process for the preparationof compound of formula (VI) of the invention comprising the step ofcoupling compound of formula (14), compound of formula (14)′ andcompound of formula (12) to give compound of formula (VI),

wherein LG is a leaving group; R₆, W, X, Y, X′, Y′, Z′ A, A′, D, D′, Land

have the same definition as above.

Another aspect of the invention provides a process for the preparationof compound of formula (VI) of the invention comprising the steps of:

a) converting compound of formula (19) into aldehyde of formula (20);and

b) converting compound of formula (20) into compound of formula (VI),

wherein W′ is COOR or CH₂OW″, wherein R has the same definition as aboveand W″ is a protecting group; R₆, W, X, Y, X′, Y′, Z′ A, A′, D, D′, Land

have the same definition as above.In Vitro Cytotoxicity of Compounds

The in vitro cytotoxicity of the cytotoxic compounds (e.g.,indolinobenzodiazepine or oxazolidinobenzodiazepine), derivativesthereof, dimers thereof or conjugates thereof of the present inventioncan be evaluated for their ability to suppress proliferation of variouscancerous cell lines in vitro (Tables 1, 2 in FIGS. 31, 32.). Forexample, cell lines such as the human breast carcinoma line SK-Br-3, orthe human epidermoid carcinoma cell line KB, can be used for theassessment of cytotoxicity of these new compounds. Cells to be evaluatedcan be exposed to the compounds for 72 hours and the surviving fractionsof cells measured in direct assays by known methods. IC₅₀ values canthen be calculated from the results of the assays.

Examples of in vitro cytotoxicity of compounds of the present inventionthat were tested on a panel of cancer cell lines and their data is shownin Table 1. All the indolinobenzodiazepine dimer compounds tested werehighly potent with IC₅₀ values in the low picomolar range. IGN-09retained most of its potency on multi-drug resistant cell lines such asCOLO205-MDR (only 4-fold higher IC₅₀ than COLO205). Compounds of theinvention are 1000 to 10,000-fold more cytotoxic than other DNAinteracting drugs used in cancer treatment, such as doxorubicin,melphalan and cis-platin. In a direct comparison, the potency of thenon-linker bearing compounds IGN1 (compound 18) and IGN09 (compound 15)was compared to the linker-bearing compounds IGN03 (compound 34) andIGN05 (compound 36) was tested towards a representative cell line Ramos.As shown in Table 2, all four compounds are highly potent with IC₅₀values less than 1 picomolar, demonstrating that the incorporation oflinker does not affect potency.

Cell-Binding Agents

The effectiveness of the compounds (e.g., indolinobenzodiazepine oroxazolidinobenzodiazepine), derivatives thereof, dimers thereof orconjugates thereof of the invention as therapeutic agents depends on thecareful selection of an appropriate cell-binding agent. Cell-bindingagents may be of any kind presently known, or that become known andincludes peptides and non-peptides. Generally, these can be antibodies(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell-binding molecule or substance.

More specific examples of cell-binding agents that can be used include:

polyclonal antibodies;

monoclonal antibodies;

fragments of antibodies such as Fab, Fab′, and F(ab′)₂, Fv (Parham, J.Immunol. 131:2895-2902 (1983); Spring et al. J. Immunol. 113:470-478(1974); Nisonoff et al. Arch. Biochem. Biophys. 89:230-244 (1960));

interferons (e.g. alpha., .beta., .gamma.);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyrotropin releasing hormone), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens;

growth factors and colony-stimulating factors such as EGF, TGF-alpha,FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158(1984));

transferrin (O'Keefe et al. J. Biol. Chem. 260:932-937 (1985)); and

vitamins, such as folate.

Monoclonal antibody techniques allow for the production of extremelyspecific cell-binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.Sensitized human cells can also be used. Another method of creatingmonoclonal antibodies is the use of phage libraries of scFv (singlechain variable region), specifically human scFv (see e.g., Griffiths etal., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO92/01047; Liming et al., WO 99/06587). In addition, resurfacedantibodies disclosed in U.S. Pat. No. 5,639,641 may also be used, as maychimeric antiobodies and humanized antibodies. Selection of theappropriate cell-binding agent is a matter of choice that depends uponthe particular cell population that is to be targeted, but in generalhuman monoclonal antibodies are preferred if an appropriate one isavailable.

For example, the monoclonal antibody MY9 is a murine IgG₁ antibody thatbinds specifically to the CD33 Antigen {J. D. Griffin et al 8 LeukemiaRes., 521 (1984)} and can be used if the target cells express CD33 as inthe disease of acute myelogenous leukemia (AML). Similarly, themonoclonal antibody anti-B4 is a murine IgG₁, that binds to the CD19antigen on B cells {Nadler et al, 131 J. Immunol. 244-250 (1983)} andcan be used if the target cells are B cells or diseased cells thatexpress this antigen such as in non-Hodgkin's lymphoma or chroniclymphoblastic leukemia HuB4 is a resurfaced antibody derived from themurine anti-B4 antibody (Roguska et al., 1994, Proc. Natl. Acad. Sci.,91, pg 969-973). HuN901 is a humanized antibody that binds to the CD56antigen expressed on small cell lung cancer, multiple myeloma, ovariancancer and other solid tumors including neuroendocrine cancers (Roguskaet al., 1994, Proc. Natl. Acad. Sci., 91, pg 969-973). B38.1 is achimeric antibody targeting EpCAM. Fully human antibodies such aspanitumumab targeting the EGF receptor expressed on several solid tumorsmay also be used (Van Cutsem et al., J Clin Oncol. 2007;25(13):1658-1664). The cell-binding agent that comprises the conjugatesand the modified cell-binding agents of the present invention may be ofany kind presently known, or that become known, and includes peptidesand non-peptides. The cell-binding agent may be any compound that canbind a cell, either in a specific or non-specific manner. Generally,these can be antibodies (especially monoclonal antibodies and antibodyfragments), interferons, lymphokines, hormones, growth factors,vitamins, nutrient-transport molecules (such as transferrin), or anyother cell-binding molecule or substance.

Where the cell-binding agent is an antibody, it binds to an antigen thatis a polypeptide and may be a transmembrane molecule (e.g. receptor) ora ligand such as a growth factor. Exemplary antigens include moleculessuch as renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor vmc, factor IX, tissue factor (TF), and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin, such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins, EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2,EphA receptors, EphB receptors, folate receptor, FOLR1, mesothelin,cripto, alpha_(v)beta₆, integrins, VEGF, VEGFR, tarnsferrin receptor,IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4,CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28,CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70,CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CD152 or anantibody which binds to one or more tumor-associated antigens orcell-surface receptors disclosed in US Publication No. 20080171040 or USPublication No. 20080305044 and are incorporated in their entirety byreference; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon, such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe HIV envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

Additionally, GM-CSF, which binds to myeloid cells can be used as acell-binding agent to diseased cells from acute myelogenous leukemia.IL-2 which binds to activated T-cells can be used for prevention oftransplant graft rejection, for therapy and prevention ofgraft-versus-host disease, and for treatment of acute T-cell leukemia.MSH, which binds to melanocytes, can be used for the treatment ofmelanoma. Folic acid can be used to target the folate receptor expressedon ovarian and other tumors. Epidermal growth factor can be used totarget squamous cancers such as lung and head and neck. Somatostatin canbe used to target neuroblastomas and other tumor types.

Cancers of the breast and testes can be successfully targeted withestrogen (or estrogen analogues) or androgen (or androgen analogues)respectively as cell-binding agents.

Production of Cytotoxic Conjugates

The present invention also provides cytotoxic compound-cell-bindingagent conjugates comprising a cell binding agent linked to one or morecytotoxic compounds via a variety of linkers, including, but not limitedto, disulfide linkers, thioether linkers, amide bonded linkers,peptidase labile linkers, acid-labile linkers, esterase-labile linkers.Representational cytotoxic conjugates of the invention areantibody/cytotoxic compound, antibody fragment/cytotoxic compound,epidermal growth factor (EGF)/cytotoxic compound, melanocyte stimulatinghormone (MSH)/cytotoxic compound, thyroid stimulating hormone(TSH)/cytotoxic compound, somatostatin/cytotoxic compound,folate/cytotoxic compound, estrogen/cytotoxic compound, estrogenanalogue/cytotoxic compound, androgen/cytotoxic compound, and androgenanalogue/cytotoxic compound.

In a preferred embodiment, the present invention provides anindolinobenzodiazepine dimer-cell-binding agent conjugate comprising thecytotoxic agent and the cell binding agent linked through a covalentbond. The linker can be cleaved at the site of the tumor/unwantedproliferating cells to deliver the cytotoxic agent to its target in anumber of ways. The linker can be cleaved, for example, by low pH(hydrazone), reductive environment (disulfide), proteolysis(amide/peptide link), or through an enzymatic reaction(esterase/glycosidase).

In a preferred aspect, representatative cytotoxic conjugates of theinvention are antibody/indolinobenzodiazepine dimer, antibodyfragment/indolinobenzodiazepine dimer, epidermal growth factor(EGF)/indolinobenzodiazepine dimer, melanocyte stimulating hormone(MSH)/indolinobenzodiazepine dimer, thyroid stimulating hormone(TSH)/indolinobenzodiazepine dimer, somatostatin/indolinobenzodiazepinedimer, folate/indolinobenzodiazepine dimer,estrogen/indolinobenzodiazepine dimer, estrogenanalogue/indolinobenzodiazepine dimer, prostate specific membraneantigen (PSMA) inhibitor/indolinobenzodiazepine dimer, matriptaseinhibitor/indolinobenzodiazepine dimer, designed ankyrin repeat proteins(DARPins)/indolinobenzodiazepine dimer, androgen/indolinobenzodiazepinedimer, and androgen analogue/indolinobenzodiazepine dimer.

Disulfide containing cytotoxic conjugates can be made by reacting athiol-containing cytotoxic agent such as 49 with an appropriatelymodified cell-binding agent. These conjugates may be purified to removenon-linked cytotoxic agent by using gel-filtration, ion exchangechromatography, ceramic hydroxyappetite (CHT) chromatography,hydrophobic interaction chromatography (CHT), tangential flow filtration(TFF), or by HPLC.

A solution of an antibody in aqueous buffer may be incubated with amolar excess of an antibody modifying agent such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or withN-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) to introducedithiopyridyl groups. The modified antibody is then reacted with thethiol-containing cytotoxic agent such as compound 49 to produce adisulfide-linked antibody-indolinobenzodiazepine dimer conjugate. Thecytotoxic-cell binding conjugate may then be purified using any of theabove mentioned methods.

Alternatively, the antibody may be incubated with a molar excess of anantibody modifying agent such as 2-iminothiolane, L-homocysteinethiolactone (or derivatives), or N-Succinimidyl-5-acetylthioacetate(SATA) to introduce sulfhydryl groups. The modified antibody is thenreacted with the appropriate disulfide-containing cytotoxic agent, suchas, compound 51 to produce a disulfide-linked antibody-cytotoxic agentconjugate. The antibody-cytotoxic agent conjugate may then be purifiedby gel-filtration or other methods mentioned above.

The number of cytotoxic molecules bound per antibody molecule can bedetermined spectrophotometrically by measuring the ratio of theabsorbance at 280 nm and 330 nm. An average of 1-10 cytotoxicmolecules/antibody molecule(s) can be linked by this method. Thepreferred average number of linked cytotoxic molecules per antibodymolecule is 2-5, and the most preferred is 3-4.5.

Alternatively, a solution of an antibody in aqueous buffer may beincubated with a molar excess of an antibody-modifying agent such asN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate tointroduce maleimido groups, or withN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) to introduceiodoacetyl groups. The modified antibody is then reacted with thethiol-containing cytotoxic agent to produce a thioether-linkedantibody-cytotoxic conjugate. The antibody-cytotoxic conjugate may thenbe purified by gel-filtration or other methods mentioned above or bymethods known to one of skill in the art.

Cytotoxic agents containing linkers terminating in an N-Hydroxysuccinimidyl (NHS) ester, such as compounds 43, 44, and 46, can bereacted with the antibody to produce direct amide linked conjugates suchas huN901-IGN-03 and huN901-IGN-07. The antibody-cytotoxic agentconjugate may then be purified by gel-filtration or other methodsmentioned above.

The following cell-binding agent/cytotoxic agent conjugates can beprepared using the appropriate linkers. Dimer 1 and 2 with peptidecleavable linkers can be prepared from the corresponding NHS esters,Dimer 3 can be made by reacting the appropriate thiol-containingcytotoxic agent with SMCC modified cell binding agent, and acid-labilehydrazone Dimer 4 can be prepared through condensation of a cytotoxicagent containing an alkyl, aryl ketone with a hydrazide modified cellbinding agent.

Asymmetric indolinobenzodiazepine dimer conjugates such as Dimers 5-8can also be prepared using similar methods to those described above.

Conjugates of cell-binding agents with cytotoxic agents of the inventioncan be evaluated for their ability to suppress proliferation of variousunwanted cell lines in vitro. For example, cell lines such as the humancolon carcinoma line COLO 205, the rhabdomyosarcoma cell line RH-30, andthe multiple myeloma cell line MOLP-8 can be used for the assessment ofcytotoxicity of these conjugates. Cells to be evaluated can be exposedto the compounds for 1-5 days and the surviving fractions of cellsmeasured in direct assays by known methods. IC₅₀ values can then becalculated from the results of the assays.

Examples of in vitro potency and target specificity ofantibody-cytotoxic agent conjugates of the present invention are shownin FIG. 21-26. All of the conjugates with cytotoxic agent/antibodyratios of 1-3 are extremely cytotoxic on the antigen positive cancercells with an IC₅₀ in the low picomolar range. Antigen negative celllines remained viable when exposed to the same conjugates. The targetspecificity of conjugates of the indolinobenzodiazepine dimers are >1000with the antibodies huN901 (anti-CD56) and muB38.1 (anti-EpCAM). Forexample, the B38.1-IGN-3 conjugate killed antigen positive COLO 205cells with an IC₅₀ value of 1.86 pM, while the antigen negative Namalwacell line was about 200-fold less sensitive with an IC₅₀ value of 336.3pM, demonstrating antigen specificity. In addition, the conjugate isalso highly potent towards the multidrug resistant COLO 205 MDR cellline with an IC₅₀ value of 16 pM. Similarly, the huN901-IGN3 conjugatewas highly potent, with an IC₅₀ value of 15 pM for antigen positive RH30cells (FIG. 22). Addition of an excess of unconjugated huN901 antibodyabolished this cytotoxic effect (IC₅₀>3 nM), demonstratingantigen-specificity. Another huN901-IGN conjugate (huN901-IGN-07) alsoshowed high potency towards antigen expressing RH-30 cells, with drugload dependent cytotoxicity and IC₅₀ values of 16 pm, 3 pM and 2 pMrespectively for conjugates bearing 1.2, 2.0 and 3.0 linked drugs perantibody molecule (FIG. 23). Similar results were obtained withhuN901-IGN07 and huN901-IGN03 towards antigen-positive Molp-8 cells.Hu901-IGN07 gave IC₅₀ values of 5 pM, 3 pM and 2 pM respectively forIGN07 loads of 1.2, 2.0 and 3.0 (FIG. 24). The huN901-IGN07 and IGN03conjugates were much less potent towards antigen negative Namalwa cellswith IC₅₀ values ranging from 1000 pM to >3000 pM (FIG. 25). TheB38.1-IGN10 conjugate was also specifically potent killing antigenpositive COLO 205 cells, with an IC₅₀ of 17 pM, and less potent (170 pM)for antigen-negative Ramos cells (FIG. 26).

In one example, in vivo efficacy of a cell binding agent/cytotoxic agentconjugate was measured. Nude mice bearing human MOLP-8 tumors weretreated with huN901-IGN-07 conjugate and significant tumor regressionwas observed compared while untreated mice tumors grew rapidly (FIG.27).

The indolinobenzodiazepine dimers of the present invention bind andalkylate double-stranded DNA (dsDNA) containing guanine residues onopposite strands spaced 4 base pairs apart. FIGS. 28-30 present datafrom reverse-phase ion pair chromatography assays showing rate ofIGN-01, IGN-02, and IGN-09 binding and crosslinking to dsDNA. Theindolino group (IGN-01) is preferred to the oxazole group (IGN-02) forrapid DNA binding and interstrand crosslinking (ICL). Initial rate ofIGN-1-DNA adduct formation is dependent on DNA sequence. IGN1 bindsfaster to DNA containing an internal GATC motif than DNA with a GTACsequence. DNA probe substituted with deoxyInosine (I) (containing no C-2amino group) in place of guanine (G) showed no reaction with IGN-1 (FIG.29).

The IC₅₀ values of various compounds of the present invention towards apanel of cell lines is listed in FIG. 31. Comparative in vitro potencyof linkable and non-linkable compounds of the present invention areshown in FIG. 32. Incorporation of a linker does not significantlyaffect potency of the parent compounds.

Compositions and Methods of Use

The present invention includes a composition (e.g., a pharmaceuticalcomposition) comprising novel benzodiazepine compounds (e.g.,indolinobenzodiazepine or oxazolidinobenzodiazepine), derivativesthereof, or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier). Thepresent invention also includes a composition (e.g., a pharmaceuticalcomposition) comprising novel benzodiazepine compounds, derivativesthereof or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier), furthercomprising a second therapeutic agent. The present compositions areuseful for inhibiting abnormal cell growth or treating a proliferativedisorder in a mammal (e.g., human). The present compositions are alsouseful for treating depression, anxiety, stress, phobias, panic,dysphoria, psychiatric disorders, pain, and inflammatory diseases in amammal (e.g., human).

The present invention includes a method of inhibiting abnormal cellgrowth or treating a proliferative disorder in a mammal (e.g., human)comprising administering to said mammal a therapeutically effectiveamount of novel benzodiazepine compounds (e.g., indolinobenzodiazepineor oxazolidinobenzodiazepine), derivatives thereof, or conjugatesthereof, (and/or solvates and salts thereof) or a composition thereof,alone or in combination with a second therapeutic agent.

The present invention also provides methods of treatment comprisingadministering to a subject in need of treatment an effective amount ofany of the conjugates described above.

Similarly, the present invention provides a method for inducing celldeath in selected cell populations comprising contacting target cells ortissue containing target cells with an effective amount of a cytotoxicagent comprising any of the cytotoxic compound-cell-binding agents(e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine dimer linkedto a cell binding agent) of the present invention, a salt or solvatethereof. The target cells are cells to which the cell-binding agent canbind.

If desired, other active agents, such as other anti-tumor agents, may beadministered along with the conjugate.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of ordinary skill in theart as the clinical situation warrants.

Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing or notcontaining about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also containan antioxidant such as tryptamine and a stabilizing agent such as Tween20.

The method for inducing cell death in selected cell populations can bepracticed in vitro, in vivo, or ex vivo.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogenic bone marrow ortissue prior to transplant in order to prevent GVHD. Treatment can becarried out as follows. Bone marrow is harvested from the patient orother individual and then incubated in medium containing serum to whichis added the cytotoxic agent of the invention, concentrations range fromabout 10 μM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e.,the dose, are readily determined by one of ordinary skill in the art.After incubation the bone marrow cells are washed with medium containingserum and returned to the patient intravenously according to knownmethods. In circumstances where the patient receives other treatmentsuch as a course of ablative chemotherapy or total-body irradiationbetween the time of harvest of the marrow and reinfusion of the treatedcells, the treated marrow cells are stored frozen in liquid nitrogenusing standard medical equipment.

For clinical in vivo use, the cytotoxic agent of the invention will besupplied as a solution or a lyophilized powder that are tested forsterility and for endotoxin levels. Examples of suitable protocols ofconjugate administration are as follows. Conjugates are given weekly for4 weeks as an intravenous bolus each week. Bolus doses are given in 50to 1000 ml of normal saline to which 5 to 10 ml of human serum albumincan be added. Dosages will be 10 μg to 2000 mg per administration,intravenously (range of 100 ng to 20 mg/kg per day). After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of inducing cell death in selected cellpopulations include malignancy of any type including, for example,cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary,and lymphatic organs; autoimmune diseases, such as systemic lupus,rheumatoid arthritis, and multiple sclerosis; graft rejections, such asrenal transplant rejection, liver transplant rejection, lung transplantrejection, cardiac transplant rejection, and bone marrow transplantrejection; graft versus host disease; viral infections, such as CMVinfection, HIV infection, AIDS, etc.; and parasite infections, such asgiardiasis, amoebiasis, schistosomiasis, and others as determined by oneof ordinary skill in the art.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (PDR). The PDR disclosesdosages of the agents that have been used in treatment of variouscancers. The dosing regimen and dosages of these aforementionedchemotherapeutic drugs that are therapeutically effective will depend onthe particular cancer being treated, the extent of the disease and otherfactors familiar to the physician of skill in the art and can bedetermined by the physician. The contents of the PDR are expresslyincorporated herein in its entirety by reference. One of skill in theart can review the PDR, using one or more of the following parameters,to determine dosing regimen and dosages of the chemotherapeutic agentsand conjugates that can be used in accordance with the teachings of thisinvention. These parameters include:

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Analogues and Derivatives

One skilled in the art of cytotoxic agents will readily understand thateach of the cytotoxic agents described herein can be modified in such amanner that the resulting compound still retains the specificity and/oractivity of the starting compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thecytotoxic agents described herein. Thus, the cytotoxic agents of thepresent invention include analogues and derivatives of the compoundsdescribed herein.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXAMPLES

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percents, ratios, parts, etc. areby weight. All reagents were purchased from the Aldrich Chemical Co.,New Jersey, or other commercial sources. Nuclear Magnetic Resonance (¹HNMR) spectra were acquired on a Bruker 400 MHz instrument and massspectra were acquired on a Bruker Daltonics Esquire 3000 instrumentusing electrospray ionization.

Example 1(2S)-1-[5-methoxy-2-nitro-4-(phenylmethoxy)-benzoyl]-2-indolinecarboxylicacid methyl ester 5

To a stirred solution of 4-benzyloxy-5-methoxy-2-nitrobenzoic acid 3(7.01 g, 23.1 mmol) in anhydrous dichloromethane (100 mL) and THF (10mL) was added oxalyl chloride (4.1 mL, 46.2 mmol) and DMF (30 μL, 0.38mmol) at room temperature. Large amounts of bubbles formed after theaddition of the DMF. The mixture was stirred overnight (the reactionusually finished within 3 hours) and then the solvents were removed byrotary evaporation in vacuo. The residue was co-evaporated one more timeby addition of anhydrous dichloromethane and high vacuumed to give theacetyl chloride 4 as a yellow solid, which was directly used for thenext step.

To a stirred solution of (s)-(−)-Indoline-2-carboxylic acid 1 (3.43 g,21.0 mmol) in anhydrous methanol (42 mL) was added thionyl chloride (3.1mL, 42.0 mmol) dropwise at 0° C. The ice bath was removed after 30minutes and the mixture continued to be stirred at room temperature for5 hours. The solvent was removed under reduced pressure and the residuewas further dried on high vacuum to give methyl ester 2, which wasdissolved in anhydrous THF ((70 mL) in a 500 mL round bottom flask. Thesolution was cooled to 0° C. and triethylamine (9.7 mL, 69.3 mmol) wasadded, followed quickly by addition of freshly prepared acetyl chloride4 in anhydrous THF (70 mL) via canula at 0° C. The mixture was stirredat 0˜5° C. for another 1.5 hours then at room temperature for 30minutes. The reaction was quenched by addition of cold 5% HCl and thendiluted with ethyl acetate and water. The aqueous layer was extractedwith ethyl acetate three times. The combined organic layers were washedsubsequently with brine, saturated sodium bicarbonate and brine, driedover anhydrous sodium sulfate and filtered. The solvents were evaporatedunder reduced pressure and the residue was purified via silica gelchromatography (Hexanes/Ethyl acetate, 2:1, 1.5:1) to give(2S)-1-[5-methoxy-2-nitro-4-(phenylmethoxy)-benzoyl]-2-indolinecarboxylicacid methyl ester 5 as a yellow solid (9.1 g, y=94%). ¹H NMR (400 Hz,CDCl₃): the compound appears as three distinct rotomers. δ 8.27 (d,J=8.4 Hz, 0.3H), 7.90 (s, 0.1H), 7.82 (s, 0.6H), 7.79 (s, 0.3H),7.50-7.28 (m, 5.4H), 7.20-7.09 (m, 1.3H), 7.05 (s, 0.6H), 6.97-6.81 (m,1.6H), 6.76 (s, 0.1H), 5.85 (d, J=8.0 Hz, 0.1H), 5.70 (d, J=8.0 Hz,0.6H), 5.45-5.41 (m, 0.6H), 5.33-5.21 (m, 2.1H), 4.55 (dd, J₁=10.8 Hz,J₂=2.8 Hz, 0.3H), 3.98 (s, 1.8H), 3.94 (s, 0.9H), 3.83-3.81 (m, 2.4H),3.62 (dd, J₁=16.4 Hz, J₂=11.4 Hz, 1H), 3.56 (s, 0.9H), 3.27-3.13 (m,1H); ¹³C NMR (400 Hz, CDCl₃): 171.5, 164.7, 155.2, 154.4, 148.6, 148.3,140.3, 137.4, 135.11, 135.05, 130.5, 129.2, 128.7, 128.4, 127.9, 127.6,127.5, 126.7, 125.5, 124.8, 124.3, 123.9, 117.6, 112.4, 110.1, 109.2,108.8, 71.3, 71.2, 61.5, 60.2, 60.1, 56.7, 56.5, 52.5, 52.4, 33.6, 31.4;HRMS (ESI, m/z): calc. 463.1505 (M+H)⁺, found 463.1516.

(2S)-1-[5-methoxy-2-nitro-4-(phenylmethoxy)-benzoyl]-2-indolinealdehyde6

To a stirred solution of the methyl ester 5 (4.4 g, 9.5 mmol) inanhydrous dichloromethane (11 mL) and toluene (33 mL) was added dibal-H(19 mL, 1.0 M in toluene) dropwise via a syringe pump in 30 minutes at−78° C. The mixture continued to be stirred at −78° C. for 3 hours andTLC (hexanes/AcOEt, 1:1.5) showed that the starting material was almostconsumed. The reaction was quenched with methanol (0.4 mL) and 5% HCl(30 mL) at −78° C. Ethyl acetate (100 mL) was added and the dryice/acetone bath was removed. The mixture was stirred at roomtemperature for 30 minutes and then transferred to a reparatory funnel.The aqueous layer was extracted with AcOEt twice and the combinedorganic layers were washed with brine, saturated sodium bicarbonate andbrine, and dried over anhydrous sodium sulfate. It was filtered throughcelite and the solvents were removed under reduced pressure (temperature<35° C.). The residue was purified by flash chromatography(Hexanes/AcOEt, 1.5:1, 1:1, 1:1.5) to give the aldehyde 6 as a yellowsolid (2.85 g, y=69%). ¹H NMR (400 Hz, CDCl₃): the compound appears asthree distinct rotomers. δ 10.02 (s, 0.3H), 9.85 (s, 0.5H), 9.45 (s,0.2H), 8.32-8.31 (m, 0.2H), 7.93 (s, 0.3H), 7.83 (s, 0.5H), 7.79 (s,0.2H), 7.53-7.34 (m, 5.2H), 7.26-7.14 (m, 1.3H), 7.08 (s, 0.5H),7.01-6.94 (m, 1H), 6.91-6.82 (m, 1H), 5.78 (d, J=8.4 Hz, 0.3H), 5.71 (d,J=8.4 Hz, 0.5H), 5.52-5.48 (m, 0.5H), 5.35-5.21 (m, 2.3H), 4.53-4.50 (m,0.2H), 4.06 (s, 1.5H), 3.98 (s, 0.6H), 3.94 (s, 0.9H), 3.63-3.17 (m,2H); HRMS (ESI, m/z): calc. 433.1400 (M+H)⁺, found 433.1387.

Compound 7

To a stirred solution of aldehyde 6 (2.16 g, 5 mmol) in THF (230 mL) wasadded deioned water (150 mL) and sodium dithionite (85%, 4.61 g, 22.5mmol). The obtained slightly cloudy solution became clear after additionof another 5 mL of deioned water. The clear mixture was stirred at roomtemperature for 16 hours and 30 mL of MeOH was added. After stirring foranother 2 hours, the solvents were removed under reduced pressure (bathtemperature below 35° C.). The residue was suspended in acetonitrile andevaporated to help remove any remaining water. The obtained white solidwas further completely dried by leaving on a high vacuum for a fewhours. The residue was suspended in dichloromethane/methanol (1:1) andfiltered through celite. The flask and the solid were thoroughly washedwith dichloromethane/methanol (1:1). The filtrate was stripped underreduced pressure. The residue was dissolved in methanol (50 mL) followedby addition of acetyl chloride (1.8 mL, 25 mmol) dropwise. The mixturewas stirred at room temperature for 30 minutes and concentrated underreduced pressure (bath temperature below 35° C.) to remove half of themethanol. The remainder was quenched with saturated sodium bicarbonatefollowed by addition of dichloromethane (150 mL) and water (100 mL). Theaqueous layer was extracted with dichloromethane (2×100 mL) and thecombined organic layers were washed with brine, dried over anhydroussodium sulfate and filtered. The solvents were removed under reducedpressure and the residue was purified by silica gel chromatography(Hexanes/AcOEt, 1:1, 1:1.3, 1:1.5) to give compound 7 as a yellow solid(1.41 g, y=73%). ¹H NMR (400 Hz, CDCl₃): δ 8.26 (d, J=8.0 Hz, 1H), 7.83(d, J=4.4 Hz, 1H), 7.57 (s, 1H), 7.46-7.23 (m, 7H), 7.11-7.08 (m, 1H),6.86 (s, 1H), 5.23 (d, J=12 Hz, 1H), 5.18 (d, J=12 Hz, 1H), 4.44 (ddd,J₁=11.2 Hz, J₂=4.4 Hz, J₃=4.0 Hz, 1H), 3.97 (s, 3H), 3.67 (dd, J₁=16.4Hz, J₂=11.2 Hz, 1H), 3.46 (dd, J₁=16.4 Hz, J₂=4.0 Hz, 1H); ¹³C NMR (400Hz, CDCl₃): δ 163.8, 163.0, 150.9, 148.3, 141.96, 139.97, 136.0, 129.4,128.6, 128.1, 128.08, 127.3, 124.7, 124.69, 120.7, 116.8, 111.9, 111.3,70.8, 56.2, 54.9, 32.5; HRMS (ESI, m/z): calc. 385.1552 (M+H)⁺, found385.1592.

Indolinobenzodiazepine (IBD) Monomer 8:

To a stirred solution of the starting material 7 (1.41 g, 3.67 mmol) indichloromethane (26 mL) was added a freshly mixed solution ofmethanesulfonic acid (26 mL) in dichloromethane (52 mL) at roomtemperature. The mixture was stirred at room temperature for 1.5 hoursand diluted with dichloromethane (100 mL). The mixture was poured on ice(˜200 g)/MeOH (10 mL). The pH of the obtained solution was adjusted to 7with saturated NaHCO₃, solid NaHCO₃ and water. The mixture was separatedand the dichloromethane layer was washed with brine. The combinedaqueous layers were extracted with ethyl acetate (3×80 mL). The ethylacetate layers were combined and washed with brine. The dichloromethaneand ethyl acetate were combined, dried over anhydrous sodium sulfate andfiltered. The solvents were removed and the residue (1.26 g) waspurified by silica gel chromatography (CH₂Cl₂/MeOH, 20:1, 15:1) to givethe IBD monomer 8 as a yellow solid (1.02 g, y=95%). ¹H NMR (400 Hz,CDCl₃): δ 8.29 (d, J=8.0 Hz, 1H), 7.91 (d, J=4.8 Hz, 1H), 7.59 (s, 1H),7.32-7.28 (m, 2H), 7.13 (t, J=7.2 Hz, 1H), 6.94 (s, 1H), 6.02 (s, —OH),4.50 (dt, J₁=10.8 Hz, J₂=4.4 Hz, 1H), 4.02 (s, 3H), 3.73 (dd, J₁=16.8Hz, J₂=10.8 Hz, 1H), 3.52 (dd, J₁=16.8 Hz, J₂=3.6 Hz, 1H); HRMS (ESI,m/z): calc. 295.1083 (M+H)⁺, found 295.1076.

Example 2 (s)-(−)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic methylester 10

To a stirred solution of(s)-(−)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic acid 9 (1.75 g,6.96 mmol) in anhydrous methanol (15 mL) was added thionyl chloride(1.02 mL, 13.9 mmol) at 0° C. After 30 minutes, the ice/water bath wasremoved and the reaction mixture continued to be stirred at roomtemperature for 3.5 hours. The reaction was quenched by addition ofsaturated sodium bicarbonate and diluted with dichloromethane (100 mL)and water (50 mL). The mixture was separated and the aqueous layer wasextracted with dichloromethane (2×50 mL). The combined organic layerswere washed with brine, dried over anhydrous sodium sulfate andfiltered. The solvents were removed under reduced pressure and theresidue was purified by silica gel chromatography (Hexanes/AcOEt, 1.5:1)to give (s)-(−)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic methylester 10 as colorless oil (1.84 g, y=99%). ¹H NMR (400 Hz, CDCl₃): thecompound appears as a pair of distinct rotomers. δ 7.35 (bs, 5H),5.22-4.99 (m, 4H), 4.53-4.45 (m, 1H), 4.22-4.09 (m, 2H), 3.76 (s, 1.5H),3.65 (s, 1.5H); MS (m/z): found 288.0 (M+Na)⁺.

Compound 11

To a stirred solution of(s)-(−)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic methyl ester 10(1.04 g, 3.92 mmol) in ethyl acetate (16 mL) was added triethyl amine(1.4 mL, 10 mmol) and palladium hydroxide on carbon (20%, 267 mg, 0.337mmol). The air in the reaction flask was removed by vacuum, then ahydrogen balloon was applied and the mixture was stirred under hydrogenatmosphere at room temperature for 2 hours. To a solution of acetylchloride 4 (prepared from 1.3 g, 4.3 mmol of4-benzyloxy-5-methoxy-2-nitrobenzoic acid 2 following the proceduresdescribed above) in anhydrous THF (15 mL) was added triethyl amine (1.1mL, 7.9 mmol) at 0° C., followed by addition of the above hydrogenationreaction mixture by filtration through celite. The palladiumcatalyst/celite was washed with anhydrous THF (15 mL). The obtainedmixture was stirred at 0° C. for 3 hours. It was diluted with ethylacetate and saturated ammonium chloride. The pH of the mixture wasadjusted to 6-7 by addition of 5% hydrochloric acid. The mixture wasseparated and the aqueous layer was extracted with ethyl acetate (2×80mL). The combined organic layers were washed with brine, dried overanhydrous sodium sulfate and filtered. The solvents were removed underreduced pressure and the residue was purified by silica gelchromatography (Hexanes/AcOEt, 1:2, 1:3) to give compound 11 as a paleyellow solid (1.49 g, y=91%). ¹H NMR (400 Hz, CDCl₃): the compoundappears as a pair of distinct rotomers. δ 7.78 (s, 0.5H), 7.75 (s,0.5H), 7.48-7.37 (m, 5H), 6.97 (s, 0.5H), 6.91 (s, 0.5H), 5.39 (d, J=4.8Hz, 0.5H), 5.26-5.23 (m, 2.5H), 4.95 (dd, J1=7.2 Hz, J2=4.4 Hz, 0.5H),4.81 (d, J=3.6 Hz, 0.5H), 4.67 (d, J=3.6 Hz, 0.5H), 4.37-4.30 (m, 1H),4.25-4.11 (m, 1.5H), 4.02 (s, 1.5H), 3.97 (s, 1.5H), 3.87 (s, 1.5H),3.67 (s, 1.5H); HRMS (ESI, m/z): calc. 417.1298 (M+H)⁺, found 417.1305.

Aldehyde 12:

To a stirred solution of the methyl ester 11 (1.49 g, 3.6 mmol) inanhydrous dichloromethane (4 mL) and toluene (12 mL) was added dibal-H(6.5 mL, 1.0 M in toluene) dropwise via a syringe pump in 30 minutes at−78° C. The mixture continued to be stirred at −78° C. for 2 hours. Thereaction was quenched with methanol (146 μL, 3.6 mmol) and 5% HCl (30mL) at −78° C. Ethyl acetate (100 mL) was added and the dry ice/acetonebath was removed. The mixture was stirred at room temperature for 30minutes and then transferred to a reparatory funnel. The aqueous layerwas extracted with AcOEt twice. All the organic layers were combined,washed with brine, saturated sodium bicarbonate and brine. It was driedover anhydrous sodium sulfate and filtered through celite. The filtratewas evaporated under reduced pressure and the residue was purified bysilica gel chromatography (Hexanes/AcOEt, 1:5, 1:10) to give thealdehyde 12 as a pale yellow solid (980 mg, y=70%). ¹H NMR (400 Hz,CDCl₃): the compound appears as a pair of distinct rotomers. δ 9.83 (s,0.67H), 9.45 (s, 0.33H), 7.77 (s, 0.67H), 7.72 (s, 0.33H), 7.45-7.37 (m,5H), 6.90 (s, 1H), 5.31-5.19 (m, 3H), 4.77 (bs, 1H), 4.67-4.56 (m, 1H),4.36-3.94 (m, 5H); HRMS (ESI, m/z): calc. 387.1192 (M+H)⁺, found387.1184.

Compound 13

To a stirred solution of aldehyde 12 (154 mg, 0.4 mmol) in THF (21 mL)was added deioned water (14 mL) and sodium dithionite (85%, 369 mg, 1.8mmol). The clear mixture was stirred at room temperature for 16 hoursand 5 mL of MeOH was added. After being stirred another 2 hours, thesolvents were removed under reduced pressure (bath temperature below 35°C.). The residue was suspended in acetonitrile and evaporated to helpremove the remaining water. The obtained white solid was furthercompletely dried by leaving on a high vacuum for a few hours. Theresidue was suspended in dichloromethane/methanol (2:1) and filteredthrough celite. The flask and the solid were thoroughly washed withdichloromethane/methanol (1:1). The filtrate was stripped under reducedpressure. The residue was dissolved in methanol (5 mL) and a freshlyprepared acetyl chloride (0.15 mL)/MeOH (5 mL) solution was addedquickly. The mixture was stirred at room temperature for 30 minutes andquenched by addition of saturated sodium bicarbonate. It was dilutedwith dichloromethane and water. The two layers were separated and theaqueous layer was extracted with dichloromethane. The combineddichloromethane layers were washed with brine and dried over anhydroussodium sulfate. The solvents were removed under reduced pressure to give127 mg crude product. The aqueous layer and the washing solution werecombined and acidified to pH 2-3 with KHSO₄. It was concentrated to halfunder reduced pressure (temperature <40° C.) and extracted withdichloromethane. The combined dichloromethane was washed with saturatedsodium bicarbonate and brine, dried over anhydrous sodium sulfate. Itwas filtered and the filtrate was evaporated under reduced pressure. Theresidue was combined with above 127 mg crude product and purified bysilica gel chromatography (Hexanes/AcOEt, 1:3, 1:5, 1:8) to givecompound 13 as a colorless foam (80 mg, y=61%). ¹H NMR (400 Hz, CDCl₃):δ 7.77 (d, J=4.0 Hz, 1H), 7.52 (s, 1H), 7.46-7.28 (m, 5H), 6.88 (s, 1H),5.28 (d, J=5.2 Hz, 1H), 5.23 (d, J=12 Hz, 1H), 5.17 (d, J=12 Hz, 1H),5.05 (d, J=5.2 Hz, 1H), 4.49 (dd, J1=9.6 Hz, J2=3.2 Hz, 1H), 4.33 (dd,J1=9.6 Hz, J2=6.4 Hz, 1H), 3.96 (s, 3H), 3.83 (dd, J1=6.4 Hz, J2=3.2 Hz,1H); MS (m/z): found 361.1 (M+Na)⁺, 379.1 (M+H₂O+Na)⁺, 339.1 (M+H)⁺.

Oxazolidinobenzodiazepine (OBD) Monomer 14:

A solution of compound 13 (90 mg, 0.27 mmol) and Pd/C (10%, 90 mg) inabsolute ethanol (1.5 mL) was bubbled with argon. 1,4-Cyclohexadiene(496 μl, 5.3 mmol) was added and the argon bubble was continued for 3hours until the starting material disappeared (TLC,dichloromethane/methanol 10:1). The mixture was then filtered throughcelite and the celite was washed with methanol. The filtrate wasevaporated under reduced pressure to give 63 mg of the crude product ascolorless foam, which was purified by silica gel chromatography(dichloromethane/methanol, 20:1) to give OBD monomer 14 (55 mg, y=82%)as a white solid. ¹H NMR (400 Hz, CDCl₃): it appears as a mixture ofimine and its methyl ethers, C11(R) and C11(S) (2:3:1). δ 7.71 (bs, 1H),7.43 (s, 0.5H), 7.41 (s, 1H), 7.18 (s, 1.5H), 6.83 (s, 1H), 6.36 (s,1.5H), 6.13 (s, 0.5H), 5.25 (d, J=4.8 Hz, 0.5H), 5.22-5.20 (m, 1H), 5.14(d, J=5.2 Hz, 1.5H), 5.10 (d, J=4.8 Hz, 0.5H), 5.05 (d, J=5.2 Hz, 1.5H),5.00-4.97 (m, 1H), 4.47 (d, J=8.8 Hz, 1.5H), 4.44-4.41 (m, 1H), 4.32(apt, J=8.0 Hz, 0.5H), 4.28-4.25 (m, 1H), 4.18-4.00 (m,2×1.5H+2×0.5H=4H), 3.84 (bs, 3×1H+0.5H=3.5H), 3.76 (bs, 3×1.5H+1H=5.5H),3.73 (s, 3×0.5H=1.5H), 3.56 (dt, J1=8.8 Hz, J2=2.8 Hz, 1.5H), 3.34 (s,3×1.5H=4.5H), 3.22 (s, 3×0.5H=1.5H); MS (m/z): found 303.1 (M+MeOH+Na)⁺,271.1 (M+Na)⁺.

Example 3 Dimer 15 (IGN-09)

To a solution of IBD monomer 8 (147 mg, 0.5 mmol) and 1,3-diiodopropane(23 μl, 0.2 mmol) in anhydrous DMF (1.0 mL) was added potassiumcarbonate (111 mg, 0.8 mmol). The mixture was stirred at roomtemperature overnight (16 hours) and diluted with dichloromethane. Itwas washed with saturated ammonium chloride and brine, dried overanhydrous sodium sulfate and filtered. The filtrate was evaporated underreduced pressure and the residue was purified through preparativereverse phase HPLC (C18 column, acetonitrile/water) to give dimer 15(IGN-09) (18.9 mg, y=15%) as a white solid. ¹H NMR (400 Hz, CDCl₃): δ8.26 (d, J=8.0 Hz, 2H), 7.87 (d, J=4.4 Hz, 2H), 7.55 (s, 2H), 7.26 (s,4H), 7.12-7.08 (m, 2H), 6.88 (s, 2H), 4.45 (ddd, J1=10.8 Hz, J2=4.4 Hz,J3=4.0 Hz, 2H), 4.36-4.26 (m, 4H), 3.94 (s, 6H), 3.70 (dd, J1=16.8 Hz,J2=10.8 Hz, 2H), 3.50 (dd, J1=16.8 Hz, J2=4.0 Hz, 2H), 2.45 (p, J=6.0Hz, 2H); HRMS (ESI, m/z): calc. 629.2400 (M+H)⁺, found 629.2400.

Example 4 Dimer 18 (IGN-01)

To a stirred solution of 1,3-Benzenedimethanol 16 (11 mg, 0.08 mmol) inanhydrous dichloromethane (0.8 mL) was added triethylamine (33 μl, 0.24mmol) then methanesulfonyl chloride (16 μL, 0.21 mmol) dropwise in 15minutes at −5˜−10° C. The solution was stirred at −5˜−10° C. for another60 minutes and was quenched with ice/water, diluted with cold ethylacetate. The mixture was separated and the organic layer was washed withcold water, dried over anhydrous sodium sulfate. It was filtered and thefiltrate was evaporated by rotary evaporation in vacuo (temperature <35°C.). The residue 17 was high vacuumed for a few hours before beingdissolved in anhydrous DMF (1.5 mL). IBD monomer 7 (94 mg, 0.32 mmol),anhydrous potassium carbonate (50 mg, 0.36 mmol) and potassium iodide(27 mg, 0.16 mmol) were added subsequently. The mixture was stirred atroom temperature for 17 hours (checked by mass spectrum) and dilutedwith dichloromethane. It was washed with brine, dried over anhydroussodium sulfate and filtered. The filtrate was evaporated under reducedpressure and the residue was purified by reverse phase HPLC (C18 column,CH₃CN/H₂O, loaded column with CH₃CN/H₂O, 3:1, stirred for 30 min andcentrifuged before injection) to furnish dimer 18 (IGN-01, 6.6 mg) as awhite solid. ¹H NMR (400 Hz, CDCl₃): δ 8.21 (d, J=8.0 Hz, 2H), 7.79 (d,J=4.4 Hz, 2H), 7.51 (s, 2H), 7.46 (s, 1H), 7.36 (bs, 3H), 7.23-7.18 (m,4H), 7.06-7.03 (m, 2H), 6.79 (s, 2H), 5.20 (d, J=12.4 Hz, 2H), 5.14 (d,J=12.4 Hz, 2H), 4.41 (ddd, J1=10.8 Hz, J2=4.4 Hz, J3=4.0 Hz, 2H), 3.92(s, 6H), 3.64 (dd, J1=17.2 Hz, J2=11.2 Hz, 2H), 3.42 (dd, J1=16.8 Hz,J2=4.0 Hz, 2H); HRMS (ESI, m/z): calc. 691.2557 (M+H)⁺, found 691.2570.

Example 5 Dimer 19 (IGN-02)

To a stirred solution of 1,3-Benzenedimethanol 16 (10 mg, 0.074 mmol) inanhydrous dichloromethane (0.8 mL) was added triethylamine (31 μl, 0.22mmol) then methanesulfonyl chloride (15 μL, 0.19 mmol) dropwise in 15minutes at −5˜−10° C. The solution was stirred at −5˜−10° C. for another60 minutes and was quenched with ice/water, diluted with cold ethylacetate. The mixture was separated and the organic layer was washed withcold water, dried over anhydrous sodium sulfate. It was filtered and thefiltrate was evaporated by rotary evaporation in vacuo (temperature <35°C.). The residue 17 was high vacuumed before dissolving in anhydrous DMF(1.5 mL). OBD monomer 14 (70 mg, 0.28 mmol), anhydrous potassiumcarbonate (51 mg, 0.37 mmol) and potassium iodide (25 mg, 0.15 mmol)were added subsequently. The mixture was stirred at room temperature for17 hours (checked by mass spectrum) and diluted with dichloromethane. Itwas washed with brine, dried over anhydrous sodium sulfate and filtered.The filtrate was evaporated under reduced pressure and the residue waspurified by reverse phase HPLC (C18 column, CH₃CN/H₂O, loaded columnwith CH₃CN/H₂O, 3:1, stirred for 30 min and centrifuged beforeinjection) to furnish dimer 19 (IGN-02, 10.0 mg) as a white solid. ¹HNMR (400 Hz, CDCl₃): δ 7.75 (d, J=4.0 Hz, 2H), 7.50-7.48 (bs, 3H), 7.38(bs, 3H), 6.83 (s, 2H), 5.26 (d, J=5.2 Hz, 2H), 5.21 (d, J=14.4 Hz, 2H),5.15 (d, J=14.0 Hz, 2H), 5.03 (d, J=5.6 Hz, 2H), 4.34-4.30 (m, 2H), 3.94(s, 6H), 3.86-3.76 (m, 2H); HRMS (ESI, m/z): calc. 599.2142 (M+H)⁺,found 599.2184.

Example 6 Triol 21

To a stirred solution of dimethyl 5-hydroxyisophthalate 20 (2.1 g, 10mmol) in anhydrous THF (50 mL) was added lithium aluminum hydride (2.0 Min THF, 10 mL, 20 mmol) at −20˜−30° C. via a syringe pump in 30 minutes.The cooling bath was removed after 30 minutes and the mixture continuedto be stirred at room temperature for 4 hours. It was cooled to 0-10° C.and quenched with saturated sodium sulfate. The mixture was diluted withacetonitrile and 5% hydrochloric acid (20 mL) was added. It was stirredfor 30 minutes and dried over anhydrous sodium sulfate. The mixture wasfiltered through celite and the filtrate was evaporated under reducedpressure. The residue was purified through silica gel chromatography(Dichloromethane/Methanol, 10:1, 8:1, 5:1) to give triol 21 (1.5 g,y=99%) as a colorless oil which became white solid after stocking ¹H NMR(400 Hz, MeOD): δ 6.78, (s, 1H), 6.69 (s, 2H), 4.50 (s, 4H). ¹³C NMR(400 Hz, MeOD): δ 158.7, 144.4, 117.8, 113.8, 65.2; MS (m/z): found153.0 (M−H)⁻.

Compound 22

To a solution of triol 21 (827 mg, 5.37 mmol) and methyl 5-bromovalerate(998 mg, 5.12 mmol) in acetonitrile (40 mL) was added potassiumcarbonate (3.71 g, 26.9 mmol). The mixture was put in a 86° C. oil bathand refluxed for 6 hours. The reaction mixture was removed from the oilbath, cooled to room temperature and the solvents were evaporated underreduced pressure (temperature <35° C.). The residue was diluted withdichloromethane and filtered. The filtrate was washed with brine, driedover anhydrous sodium sulfate and filtered. The filtrate was strippedunder reduced pressure and the residue was purified through silica gelchromatography (Hexanes/Ethyl acetate, 1:2, 1:3) to give compound 22(1.15 g, y=84%) as a white solid. ¹H NMR (400 Hz, CDCl₃): δ 6.89 (s,1H), 6.80 (s, 2H), 4.62 (s, 4H), 3.98-3.95 (m, 2H), 3.67 (s, 3H),2.41-2.37 (m, 2H), 2.23 (bs, —OH×2), 1.84-1.78 (m, 4H); MS (m/z): found291.1 (M+Na)⁺.

Compound 23

Following the procedure to prepare compound 22, compound 23 (1.43 g,y=75%) was synthesized as a white solid from triol 21 (1.16 g, 7.53mmol), methyl 4-bromobutyrate (1.52 g, 8.39 mmol) and potassiumcarbonate (5.2 g, 37.6 mmol). ¹H NMR (400 Hz, CDCl₃): δ 6.90 (s, 1H),6.80 (s, 2H), 4.62 (s, 4H), 4.00 (t, J=6.0 Hz, 2H), 3.68 (s, 3H), 2.51(t, J=7.2 Hz, 2H), 2.19 (s, —OH×2), 2.13-2.06 (m, 2H); MS (m/z): found277.1 (M+Na)⁺.

Compound 24

Following the procedure to prepare compound 22, compound 24 (515 mg,y=37%) was synthesized as a white sticky solid from triol 21 (953 mg,6.19 mmol), methyl bromoacetate (587 μl, 6.19 mmol) and potassiumcarbonate (4.3 g, 31 mmol). ¹H NMR (400 Hz, CDCl₃): δ 6.95 (s, 1H), 6.81(s, 2H), 4.64 (s, —OH×2), 4.61 (s, 4H), 3.81 (s, 3H), 2.41 (s, 2H); ¹³CNMR (400 Hz, CDCl₃): δ 169.4, 158.1, 143.0, 118.5, 112.1, 65.2, 64.8,52.3; MS (m/z): found 249.0 (M+Na)⁺.

Compound 27

To a solution of 5-nitro-m-xylene-α,α′-diol 25 (1.07 g, 5.84 mmol) inmethanol (50 mL) was added Pd/C (10%, 311 mg, 0.29 mmol). Hydrogen wasintroduced to replace the air then the mixture was hydrogenated (H₂, 5psi) for 2 hours at room temperature. The solution was filtered throughcelite and the filtrate was evaporated by rotary evaporation in vacuo togive compound 26 as a white solid (900 mg, y=100%). ¹H NMR (400 Hz,MeOD): δ 6.71 (s, 1H), 6.66 (s, 2H), 4.51 (s, 4H); ¹³C NMR (400 Hz,MeOD): δ 148.9, 143.8, 116.7, 114.3, 65.5; It was dissolved in anhydrousacetonitrile (30 mL) and ethyl bromoacetate (443 μl, 4.67 mmol) andpotassium carbonate (807 mg, 5.84 mmol) were added. The mixture was putin a 86° C. oil bath and refluxed for 17 hours. The reaction mixture wasremoved from the oil bath, cooled to room temperature and diluted withdichloromethane. It was filtered through celite and the solid was washedwith dichloromethane. White precipitate appeared in the filtrate. It wascollected by filtration to give compound 27 (414 mg, y=39%) as a whitesolid. ¹H NMR (400 Hz, MeOD): δ 6.67 (s, 1H), 6.53 (s, 2H), 4.51 (s,4H), 3.94 (s, 2H), 3.73 (s, 3H); ¹³C NMR (400 Hz, MeOD): δ 174.0, 149.7,143.9, 116.2, 111.6, 65.6, 52.6, 46.5; MS (m/z): found 248.0 (M+Na)⁺.

Compound 28

To a solution of 5-nitro-m-xylene-α,α′-diol 25 (564 mg, 3.08 mmol) inmethanol (35 mL) was added Pd/C (10%, 164 mg, 0.154 mmol). Hydrogen wasintroduced to replace the air then the mixture was hydrogenated (H₂, 5psi) for 2 hours at room temperature. The solution was filtered throughcelite and the filtrate was evaporated by rotary evaporation in vacuo togive compound 26, which was dissolved in anhydrous acetonitrile (15 mL)and methyl 4-bromobutyrate (557 mg, 3.08 mmol) and potassium carbonate(426 mg, 3.08 mmol) were added. The mixture was put in a 86° C. oil bathand refluxed for 18 hours. The reaction mixture was removed from the oilbath, cooled to room temperature and diluted with dichloromethane. Itwas filtered through celite and the solid was washed withdichloromethane/acetonitrile (1:1). The filtrate was evaporated underreduced pressure and the residue was purified through silica gelchromatography (Combiflash, dichloromethane/methanol) to give compound28 (292 mg, y=37%) as a white solid. ¹H NMR (400 Hz, MeOD): δ 6.62 (s,1H), 6.55 (s, 2H), 4.50 (s, 4H), 3.65 (s, 3H), 3.13 (d, J=7.2 Hz, 2H),2.43 (d, J=7.2 Hz, 2H), 1.89 (p, J=7.2 Hz, 2H); ¹³C NMR (400 Hz, MeOD):δ 175.9, 150.5, 143.7, 115.5, 111.7, 65.7, 52.2, 44.3, 32.5, 25.8; MS(m/z): found 276.0 (M+Na)⁺.

Compound 29

To a solution of compound 27 (230 mg, 1.02 mmol) in anhydrousacetonitrile (7 mL) was added methyl iodide (70 μl, 1.12 mmol) andpotassium carbonate (155 mg, 1.12 mmol). The mixture was put in a 86° C.oil bath and refluxed for 17 hours. The reaction mixture was removedfrom the oil bath, cooled to room temperature and diluted withdichloromethane. It was filtered through celite and the solid was washedwith dichloromethane/methanol (10:1). The filtrate was evaporated underreduced pressure and the residue was purified through silica gelchromatography (Combiflash, dichloromethane/methanol) to give compound29 (98 mg, y=40%) as a white solid. ¹H NMR (400 Hz, MeOD): δ 6.70 (s,1H), 6.63 (s, 2H), 4.84 (s, 2×-OH), 4.54 (s, 4H), 4.16 (s, 2H), 3.69 (s,3H), 3.05 (s, 3H); ¹³C NMR (400 Hz, MeOD): δ 173.6, 150.9, 143.8, 115.6,111.0, 65.7, 54.9, 52.4, 39.8; MS (m/z): found 262.0 (M+Na)⁺.

Compound 30

To a solution of compound 28 (151 mg, 0.597 mmol) in anhydrousacetonitrile (4 mL) was added methyl iodide (74 μl, 1.19 mmol) andpotassium carbonate (99 mg, 0.716 mmol). The mixture was put in an 86°C. oil bath and refluxed for 17 hours. The reaction mixture was removedfrom the oil bath, cooled to room temperature and diluted withdichloromethane. It was filtered through celite and the solid was washedwith dichloromethane/methanol (10:1). The filtrate was evaporated underreduced pressure and the residue was purified through silica gelchromatography (Combiflash, dichloromethane/methanol) to give compound30 (63 mg, y=39%) as a colorless oil. ¹H NMR (400 Hz, MeOD): δ 6.67 (s,2H), 6.65 (s, 1H), 4.54 (s, 4H), 3.65 (s, 3H), 3.36 (t, J=7.2 Hz, 2H),2.92 (s, 3H), 2.36 (t, J=7.2 Hz, 1H), 1.87 (p, J=7.2 Hz, 2H); ¹³C NMR(400 Hz, MeOD): δ 175.7, 151.3, 143.7, 115.0, 111.4, 65.9, 53.0, 52.2,38.9, 32.2, 23.3; MS (m/z): found 290.0 (M+Na)⁺.

Compound 34 (IGN-03)

To a stirred solution of compound 22 (80.4 mg, 0.3 mmol) in anhydrousdichloromethane (2 mL) was added triethylamine (125 μl, 0.9 mmol) thenmethanesulfonyl chloride (60 μL, 0.78 mmol) dropwise in 15 minutes at−5˜−10° C. The solution was stirred at −5˜−10° C. for another 60 minutesand was quenched with ice/water, diluted with cold ethyl acetate. Themixture was separated and the organic layer was washed with cold water,dried over anhydrous sodium sulfate. It was filtered and the filtratewas evaporated by rotary evaporation in vacuo (temperature <35° C.). Theresidue 31 was high vacuumed before dissolving in anhydrous DMF (3 mL).IBD monomer 7 (221 mg, 0.75 mmol) and anhydrous potassium carbonate (207mg, 1.5 mmol) were added. The mixture was stirred at room temperaturefor 20 hours (checked by mass spectrum) and diluted withdichloromethane. It was washed with water and brine, dried overanhydrous sodium sulfate and filtered. The filtrate was evaporated underreduced pressure and the residue was purified through silica gelchromatography (hexanes/ethyl acetate, 1:3, 1:4, 1:6, 1:10, thenethylacetate/methanol, 10:1) to give compound 34 (169 mg, y=68%, 86%purity based on analytical reverse phase HPLC) as a yellowish solid.Fractions that contained impurities and compound 34 were also collectedand the solvents were evaporated to give 70 mg of yellowish solid. Thetwo yellowish solids were combined and further purified through reversephase HPLC (C18 column, CH₃CN/H₂O, loaded column with CH₃CN/H₂O, 3:1,stirred for 30 min and centrifuged before injection) to furnish dimer 34(IGN-03, 103 mg, y=41%) as a white solid. ¹H NMR (400 Hz, CDCl₃): δ 8.27(d, J=8.0 Hz, 2H), 7.85 (d, J=3.2 Hz, 2H), 7.58 (s, 2H), 7.29-7.24 (m,4H), 7.12-7.07 (m, 3H), 6.94 (s, 2H), 6.83 (s, 2H), 5.22 (d, J=12.8 Hz,2H), 5.16 (d, J=12.8 Hz, 2H), 4.47 (dt, J1=11.2 Hz, J2=4.4 Hz, 2H), 3.98(bs, 8H), 3.73-3.64 (m, 2H), 3.68 (s, 3H), 3.48 (dd, J1=16.8 Hz, J2=3.6Hz, 2H), 2.42-2.38 (m, 2H), 1.83-1.80 (m, 4H); HRMS (ESI, m/z): calc.821.3187 (M+H)⁺, found 821.3188.

Compound 35 (IGN-04)

Following the procedure to prepare compound 34, compound 35 (IGN-04) wassynthesized (151 mg, y=62%, 88% purity based on analytical reverse phaseHPLC) as a yellowish solid. Part of it was further purified by reversephase HPLC for ¹H NMR analysis. ¹H NMR (400 Hz, CDCl₃): δ 8.17 (d, J=8.0Hz, 2H), 7.74 (d, J=5.2 Hz, 2H), 7.48 (s, 2H), 7.20-7.15 (m, 4H),7.03-6.99 (m, 3H), 6.85 (s, 2H), 6.75 (s, 2H), 5.12 (d, J=12.8 Hz, 2H),5.06 (d, J=12.8 Hz, 2H), 4.37 (dt, J1=11.2 Hz, J2=4.4 Hz, 2H), 3.93 (t,J=6.0 Hz, 2H), 3.86 (s, 6H), 3.64-3.57 (m, 2H), 3.60 (s, 3H), 3.39 (dd,J1=16.8 Hz, J2=3.6 Hz, 2H), 2.44 (t, J=7.2 Hz, 2H), 2.02 (p, J=6.4 Hz,2H); HRMS (ESI, m/z): calc. 807.3030 (M+H)⁺, found 807.3008.

Compound 36 (IGN-05)

Following the procedure to prepare compound 34, compound 36 (IGN-05) wassynthesized (84.5 mg, y=18%) as a white solid after preparative reversephase HPLC. ¹H NMR (400 Hz, CDCl₃): δ 8.24 (d, J=8.0 Hz, 2H), 7.79 (d,J=4.4 Hz, 2H), 7.55 (s, 2H), 7.26-7.22 (m, 4H), 7.12-7.07 (m, 3H), 6.96(s, 2H), 6.81 (s, 2H), 5.18 (d, J=12.8 Hz, 2H), 5.12 (d, J=12.8 Hz, 2H),4.64 (s, 2H), 4.44 (dt, J1=10.8 Hz, J2=4.4 Hz, 2H), 3.95 (s, 6H), 3.77(s, 3H), 3.73-3.62 (m, 2H), 3.44 (dd, J1=16.8 Hz, J2=3.6 Hz, 2H); HRMS(ESI, m/z): calc. 779.2717 (M+H)⁺, found 779.2703.

Compound 39 (IGN-06)

Following the procedure to prepare compound 34, compound 39 (IGN-06) wassynthesized in 6% yield as a white solid after preparative reverse phaseHPLC. ¹H NMR (400 Hz, CDCl₃): δ 8.28 (d, J=8.0 Hz, 2H), 7.86 (d, J=4.0Hz, 2H), 7.58 (s, 2H), 7.31-7.26 (m, 4H), 7.12 (t, J=7.2 Hz, 2H),6.90-6.86 (m, 3H), 6.72 (s, 2H), 5.22 (d, J=12.4 Hz, 2H), 5.13 (d,J=12.4 Hz, 2H), 4.51-4.46 (m, 2H), 3.99 (s, 6H), 3.74-3.68 (m, 2H), 3.71(s, 3H), 3.49 (dd, J₁=16.8 Hz, J₂=3.6 Hz, 2H), 3.09 (s, 3H); HRMS (ESI,m/z): calc. 792.3033 (M+H)⁺, found 792.3013.

Compound 40 (IGN-07)

Following the procedure to prepare compound 34, compound 40 (IGN-07) wassynthesized in 21% yield as a white solid after preparative reversephase HPLC. ¹H NMR (400 Hz, CDCl₃): δ 8.27 (d, J=8.0 Hz, 2H), 7.84 (d,J=4.4 Hz, 2H), 7.58 (s, 2H), 7.30-7.23 (m, 4H), 7.21-7.02 (m, 3H), 6.88(s, 2H), 6.74 (s, 2H), 5.23-5.13 (m, 4H), 4.50-4.42 (m, 2H), 3.99 (s,6H), 3.74-3.70 (m, 2H), 3.67 (s, 3H), 3.51-3.33 (m, 4H), 2.92 (s, 3H),2.36-2.30 (m, 2H), 1.93-1.84 (m, 2H); HRMS (ESI, m/z): calc. 820.3346(M+H)⁺, found 820.3329.

Example 7 Compound 41

To a solution of compound 34 (42 mg, 0.051 mmol) in anhydrous1,2-dichloroethane (1 mL) was added trimethyltin hydroxide (139 mg, 0.77mmol). The mixture was heated at 78˜82° C. (80° C. oil bath) and stirredovernight. The TLC (CH₂Cl₂/MeOH, 10:1) showed the disappearance of thestarting material. The reaction mixture was cooled to room temperatureand diluted with dichloromethane. It was washed with drops of 5%hydrochloric acid in brine, saturated ammonium chloride and brine, driedover anhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography (combiflash, CH₂Cl₂/MeOH, from 1:0to 5:1) to give IGN-03 acid 41 (33.8 mg, y=82%) as a yellowish solid.The residue can also be used for next step without purification. MS(m/z): found 805.1 (M−H)⁻, 823.0 (M+H₂O—H)⁻, 829.2 (M+Na)⁺, 847.2(M+H₂O+Na)⁺.

Compound 42

To a stirred solution of compound 35 (32 mg, 0.040 mmol) in a mixture ofTHF (0.4 mL), methanol (0.1 mL) and deioned water (0.1 ml) was addedfreshly prepared 2N LiOH (24 μl, 0.048 mmol) at 0° C. The cooling bathwas removed and the mixture was stirred at room temperature for 8 hours.The reaction mixture was diluted with ethyl acetate and water. The pH ofthe mixture was adjusted to 4˜5 with 5% hydrochloric acid. It was washedwith brine, dried over anhydrous sodium sulfate and filtered. Thefiltrate was evaporated under reduced pressure and the residue waspurified by preparative reverse phase HPLC (C18 column,acetonitrile/H₂O) to give the IGN-04 acid 42 (4.2 mg, y=13%) as a whitesolid. MS (m/z): found 791.0 (M−H)⁻, 809.0 (M+H₂O—H)⁻, 815.2 (M+Na)⁺,833.1 (M+H₂O+Na)⁺.

Compound 43

To a stirred solution of IGN-03 acid 41 (8.9 mg, 0.011 mmol) inanhydrous dichloromethane (0.2 mL) was added N-hydroxysuccinimide (2.6mg, 0.022 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (4.2 mg, 0.022 mmol) and a tiny particle ofdimethylaminopyridine. The mixture was stirred at room temperatureovernight and diluted with dichloromethane. It was washed with saturatedammonium chloride and brine, dried over anhydrous sodium sulfate andfiltered. The filtrate was evaporated under reduced pressure. Theresidue was purified through silica gel chromatography (Combiflash,CH₂Cl₂/MeOH, from 1:0 to 10:1) to give IGN-03 NHS eater 43 (7.9 mg,y=79%) as a yellowish solid. Reverse phase preparative HPLC (C18 column,CH₃CN/H₂O, extracted the product fractions with dichloromethane)purification gave 3.2 mg white solid for ¹H NMR analysis. ¹H NMR (400Hz, CDCl₃): δ 8.28 (d, J=8.0 Hz, 2H), 7.87 (d, J=4.0 Hz, 2H), 7.59 (s,2H), 7.31-7.27 (m, 4H), 7.15-7.10 (m, 3H), 6.97 (s, 2H), 6.86 (s, 2H),5.25 (d, J=12.4 Hz, 2H), 5.18 (d, J=12.4 Hz, 2H), 4.49 (dt, J₁=10.8 Hz,J₂=4.0 Hz, 2H), 4.04 (t, J=5.6 Hz, 2H), 4.01 (s, 6H), 3.72 (dd, J₁=16.8Hz, J₂=10.8 Hz, 2H), 3.51 (dd, J₁=16.8 Hz, J₂=4.0 Hz, 2H), 2.85 (bs,4H), 2.72 (t, J=6.8 Hz, 2H), 1.99-1.91 (m, 4H); HRMS (ESI, m/z): calc.904.3194 (M+H)⁺, found 904.3182.

Compound 44

Following the procedure to prepare compound 43, compound 44 wassynthesized in 86% yield as a yellowish solid. MS (m/z): found 944.2(M+MeOH+Na)⁺, 976.2 (M+2MeOH+Na)⁺.

Compound 45 (IGN-07 Acid)

To a solution of compound 40 (14 mg, 0.017 mmol) in anhydrous1,2-dichloroethane (0.5 mL) was added trimethyltin hydroxide (62 mg,0.34 mmol). The mixture was heated at 78˜82° C. (80° C. oil bath) andstirred overnight. The TLC (CH₂Cl₂/MeOH, 10:1) showed the disappearanceof the starting material. The reaction mixture was cooled to roomtemperature and diluted with dichloromethane. It was washed withsaturated ammonium chloride and brine, dried over anhydrous sodiumsulfate, filtered and evaporated to give IGN-07 acid 45 as a paleyellowish solid (29.2 mg, contaminated with trimethyltin hydroxide). MS(m/z): found 804.1 (M−H)⁻, 822.1 (M+H₂O—H)⁻, 828.2 (M+Na)⁺, 846.2(M+H₂O+Na)⁺. It was used for next step without purification.

Compound 46

To a stirred solution of IGN-07 acid 45 from above reaction (0.017 mmol)in anhydrous dichloromethane (0.5 mL) was added N-hydroxysuccinimide(6.1 mg, 0.051 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (9.8 mg, 0.051 mmol) and a tiny particle ofdimethylaminopyridine. The mixture was stirred at room temperatureovernight and diluted with dichloromethane. It was washed with saturatedammonium chloride and brine, dried over anhydrous sodium sulfate andfiltered. The filtrate was evaporated under reduced pressure. Theresidue was purified through silica gel chromatography (Combiflash,CH₂Cl₂/MeOH, from 1:0 to 10:1) to give IGN-07 NHS eater 46 (9.1 mg,y=59% for two steps from IGN-07) as a yellowish solid. ¹H NMR (400 Hz,CDCl₃): δ 8.25 (d, J=7.6 Hz, 2H), 7.82 (d, J=4.4 Hz, 2H), 7.55 (s, 2H),7.26-7.18 (m, 5H), 7.09 (t, J=7.6 Hz, 2H), 6.84 (s, 2H), 6.74 (s, 2H),5.21 (d, J=12.4 Hz, 2H), 5.15 (d, J=12.4 Hz, 2H), 4.46-4.42 (m, 2H),3.98 (s, 6H), 3.72-3.64 (m, 2H), 3.44-3.37 (m, 4H), 2.95 (s, 3H), 2.74(bs, 4H), 2.57 (t, J=7.2 Hz, 2H), 1.95 (t, J=7.2 Hz, 2H); HRMS (ESI,m/z): calc. 903.3354 (M+H)⁺, found 903.3347.

Example 8 Compound 47

To a stirred solution of cysteamine hydrochloride (568 mg, 5 mmol) inanhydrous methanol (15 mL) was added S-methyl methanethiosulfonate (519μl, 5.5 mmol) at 0° C. The mixture was stirred at room temperatureovernight. Triethylamine (1.4 mL, 10 mmol) was added and the solventswere removed under reduced pressure. The residue was dissolved in 50 mLof anhydrous dichloromethane and gave a 0.1 M solution of compound 47 indichloromethane (assuming 100% yield). An aliquot of the solution (0.2mL) was used for next step reaction. The rest of the solution wasdiluted with dichloromethane, washed with saturated sodium bicarbonateand brine, dried over anhydrous sodium sulfate and filtered. Thefiltrate was evaporated under reduced pressure and the residue waspurified through silica gel chromatography (dichloromethane/methanol,10:1 with 1% triethylamine) to give compound 47 (82 mg, y=13%, productlost in the aqueous work up due to its good water solubility) as acolorless oil. ¹H NMR (400 Hz, CDCl₃): δ 3.02 (t, J=6.4 Hz, 2H), 2.77(t, J=6.4 Hz, 2H), 2.41 (s, 3H), 1.34 (bs, 2H).

Compound 48 (IGN-08)

To a flask containing IGN-03 acid 41 (8.1 mg, 0.01 mmol) was added above0.1 M solution of compound 47 in anhydrous dichloromethane (0.2 mL).N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.8 mg,0.02 mmol), triethylamine (1.4 μl, 0.01 mmol) and a tiny particle ofdimethylaminopyridine were added. The mixture was stirred at roomtemperature overnight and diluted with dichloromethane. It was washedwith saturated ammonium chloride and brine, dried over anhydrous sodiumsulfate and filtered. The filtrate was evaporated under reduced pressureand the residue was purified through preparative reverse phase HPLC (C18column, acetonitrile/H₂O) to give compound 48 (4.0 mg, y=44%) as a whitesolid. ¹H NMR (400 Hz, CDCl₃): δ 8.25 (d, J=8.0 Hz, 2H), 7.84 (d, J=4.4Hz, 2H), 7.57 (s, 2H), 7.29-7.24 (m, 4H), 7.10 (t, J=7.6 Hz, 2H), 7.06(s, 1H), 6.92 (s, 2H), 6.82 (s, 2H), 5.22 (d, J=12.8 Hz, 2H), 5.17 (d,J=12.4 Hz, 2H), 4.46 (dt, J₁=11.2 Hz, J₂=4.4 Hz, 2H), 3.98 (bs, 8H),3.69 (dd, J₁=16.8 Hz, J₂=10.8 Hz, 2H), 3.62 (d, J=6.4 Hz, 1H), 3.58 (d,J=6.0 Hz, 1H), 3.48 (dd, J₁=17.2 Hz, J₂=3.6 Hz, 2H), 2.82 (t, J=6.4 Hz,2H), 2.39 (s, 3H), 2.23 (t, J=6.8 Hz, 2H), 1.80-1.78 (m, 4H); HRMS (ESI,m/z): calc. 912.3101 (M+H)⁺, found 912.3118.

Compound 49

To a suspension of tris(2-carboxyethyl) phosphine hydrochloride(TCEP.HCl, 3.8 mg, 0.013 mmol) in a drop of deioned water (˜50 μL) wasadded saturated sodium bicarbonate dropwise (˜25 μL) to adjust the pH toabout 6˜7, followed by addition of pH 6.5 buffer solution (0.1 Mphosphate buffer, 0.3 mL). The obtained mixture was added to thesolution of compound 48 (IGN-08, 4.0 mg, 0.0044 mmol) in methanol (1.0mL) and acetonitrile (1.0 mL). The solution was stirred at roomtemperature for 1.5 hours and diluted with pH 6.5 buffer anddichloromethane (the reaction was checked by mass spectra, which showedonly the product signals). It was separated and the organic layer waswashed with brine, dried over anhydrous sodium sulfate and filtered. Thefiltrate was evaporated under reduced pressure and the residue waspurified by silica gel chromatography (Combiflash, dichloromethane/MeOH)to give product 49 as a pale yellow solid (2.7 mg, y=71%). MS (m/z):found 864.0 (M−H)⁻, 932.0 (M+MeOH+2H₂O—H)⁻, 888.1 (M+Na)⁺, 920.2(M+MeOH+Na)⁺, 952.2 (M+2MeOH+Na)⁺.

Example 9 Compound 50

To a stirred solution of cysteamine hydrochloride (227 mg, 2 mmol) inanhydrous methanol (10 mL) was added aldrithiol (661 mg, 3 mmol).Reaction solution became clear yellow from clear colorless after theaddition of aldrithiol. The mixture was stirred at room temperature for21 hours. Triethylamine (279 μl, 2 mmol) was added and the solvents wereremoved under reduced pressure. The residue was purified through silicagel chromatography (Combiflash, dichloromethane/methanol, 1:0 to 15:1with 0.1% triethylamine) to give compound 50 (301 mg, y=81%) as acolorless oil. ¹H NMR (400 Hz, CDCl₃): δ 8.52-8.49 (m, 1H), 7.69-7.60(m, 2H), 7.15-7.10 (m, 1H), 3.04 (t, J=6.0 Hz, 2H), 2.92 (t, J=6.0 Hz),1.92 (bs, 2H).

Compound 51 (IGN-10)

To a solution of IGN-03 acid 41 (from 0.05 mmol of IGN-03 withoutpurification) in anhydrous dichloromethane (1 mL) was added compound 50(37 mg, 0.2 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (38 mg, 0.2 mmol) and a tiny particle ofdimethylaminopyridine. The mixture was stirred at room temperatureovernight and diluted with dichloromethane. It was washed with saturatedammonium chloride and brine, dried over anhydrous sodium sulfate andfiltered. The filtrate was evaporated under reduced pressure. Theresidue was purified through silica gel chromatography (Combiflash,dichloromethane/methanol, 1:0 to 5:1) to give 51 mg of yellow foam,which was further purified through preparative reverse phase HPLC (C18column, acetonitrile/H₂O) to give compound 51 (7.4 mg, y=15%) as ayellowish solid. ¹H NMR (400 Hz, CDCl₃): δ 8.50 (d, J=4.4 Hz, 1H), 8.28(d, J=8.0 Hz, 2H), 7.87 (d, J=4.4 Hz, 2H), 7.63-7.59 (m, 3H), 7.52 (d,J=8.0 Hz, 1H), 7.31-7.21 (m, 4H), 7.14-7.09 (m, 4H), 6.96 (s, 2H), 6.85(s, 2H), 5.23 (d, J=12.8 Hz, 2H), 5.18 (d, J=12.4 Hz, 2H), 4.49 (dt,J₁=11.2 Hz, J₂=4.4 Hz, 2H), 4.03-4.00 (m, 8H), 3.72 (dd, J₁=16.8 Hz,J₂=11.2 Hz, 2H), 3.60 (d, J=5.6 Hz, 1H), 3.57 (d, J=5.6 Hz, 1H), 3.50(dd, J₁=16.8 Hz, J₂=3.6 Hz, 2H), 2.95 (t, J=5.6 Hz, 2H), 2.30 (t, J=6.4Hz, 2H), 1.85-1.84 (m, 4H); HRMS (ESI, m/z): calc. 975.3210 (M+H)⁺,found 975.3190.

Compound 53

To a stirred solution of 4-benzyloxy-3-methoxybenzyl alcohol 52 (2.5 g,10 mmol) in acetic anhydride (30 mL) was added copper(II) nitratehydrate (2.7 g, 11 mmol) slowly in portion at 0° C. The obtainedsuspension continued to be stirred at 0° C. for 1 hour and at roomtemperature for 3 hours. The reaction mixture was poured on ice/waterand stirred for 1 hour. It was filtered to collect the yellow solid,which was subsequently dissolved in MeOH/THF (1:1, V/V, 30 mL).Potassium carbonate (2.1 g, 15 mmol) was added and the obtained mixturewas stirred at room temperature for 3 hours. It was concentrated underreduced pressure and the residue was diluted with dichloromethane,washed with water and brine, dried over anhydrous sodium sulfate andfiltered. The filtrate was evaporated under reduced pressure and theresidue was purified through silica gel chromatography (CH₂Cl₂/MeOH,20:1, 18:1, 15:1) to give compound 53 (1.50 g, y=52%) as yellow solid.¹H NMR (400 Hz, CDCl₃): δ 7.78 (s, 1H), 7.48-7.33 (m, 5H), 7.20 (s, 1H),5.18 (s, 2H), 4.96 (s, 2H), 4.01 (s, 3H).

Example 10 Compound 123

To a stirred solution of compound 122 (137 mg, 0.22 mmol) in THF (1.5mL) and MeOH (0.5 mL) was added a solution of lithium hydroxidemonohydrate (46 mg, 1.1 mmol) in deioned water (0.5 mL). The mixture wasstirred in a 60° C. oil bath for 6 hours. It was cooled to roomtemperature and diluted with ethyl acetate and water. The pH wasadjusted to 4˜5 with 5% hydrochloric acid. The aqueous layer wasextracted with ethyl acetate. The combined organic layers were washedwith saturated sodium bicarbonate and brine, dried over anhydrous sodiumsulfate and filtered. The filtrate was striped to give compound 123(87.5 mg, y=65%). MS (m/z): found 606.1 (M−H)⁻.

Compound 124

To a solution of acid 123 (87.5 mg, 0.14 mmol) in anhydrous DMF (1 mL)was added DMAP (21 mg, 0.17 mmol), methyl 5-aminovalerate hydrochloride(26 mg, 0.15 mmol) and EDC (40 mg, 0.21 mmol). The mixture was stirredat room temperature overnight and diluted with ethyl acetate. It waswashed with saturated ammonium chloride, brine, saturated sodiumbicarbonate and brine, dried over anhydrous sodium sulfate and filtered.The filtrate was striped and the residue was purified through silica gelchromatography (Combiflash, dichloromethane/MeOH) to give compound 124(71 mg, y=70%) as a yellow foam. ¹H NMR (400 Hz, CDCl₃): δ 9.07 (s, 1H),8.62 (s, 1H), 8.40 (s, 1H), 7.19 (s, 1H), 7.17 (s, 1H), 7.09 (s, 1H),7.00 (s, 1H), 6.74 (s, 1H), 6.62 (s, 3H), 6.46 (s, 1H), 3.94 (s, 3H),3.85 (bs, 12H), 3.34-3.31 (m, 2H), 2.32 (t, J=7.2 Hz, 2H), 1.68-1.55 (m,4H), 1.48 (s, 9H); MS (ESI, m/z): found 721.0 (M+H)⁺.

Compound 125

To a solution of IBD monomer 8 (118 mg, 0.4 mmol) and methyl4-bromobutyrate (109 mg, 0.6 mmol) in anhydrous DMF (1.5 mL) was addedpotassium carbonate (111 mg, 0.8 mmol). The mixture was stirred at roomtemperature overnight and diluted with ethyl acetate, washed withsaturated ammonium chloride and brine. It was dried over anhydroussodium sulfate and filtered. The filtrate was striped under reducedpressure to give compound 125 (146 mg, y=93%) as a yellow foam. ¹H NMR(400 Hz, CDCl₃): δ 8.25 (d, J=8.0 Hz, 1H), 7.84 (d, J=4.4 Hz, 1H), 7.52(s, 1H), 7.26-7.22 (m, 2H), 7.10-7.06 (m, 1H), 6.81 (s, 1H), 4.44 (dt,J₁=10.8 Hz, J₂=4.0 Hz, 1H), 4.15-4.07 (m, 2H), 3.92 (s, 3H), 3.68 (s,3H), 3.67-3.64 (m, 1H), 3.46-3.43 (m, 1H), 2.55 (t, J=7.2 Hz, 2H),2.22-2.15 (m, 2H); MS (ESI, m/z): found 465.2 (M+MeOH+K)⁺.

Compound 126

The mixture of compound 125 (146 mg, 0.37 mmol) and trimethyltinhydroxide (669 mg, 3.7 mmol) in anhydrous 1,2-dichloroethane (2 mL) washeated to 80° C. (oil bath temperature) and stirred at that temperaturefor 18 hours. The oil bath was removed and the mixture was diluted withdichloromethane, washed with brine/5% HCl (0.5 mL), saturated sodiumbicarbonate and brine, dried over anhydrous sodium sulfate and filtered.The filtrate was striped and the residue was purified through silica gelchromatography (Combiflash, dichloromethane/MeOH) to give compound 126(90 mg, y=64%) as a yellow solid. ¹H NMR (400 Hz, CDCl₃): δ 8.26 (d,J=8.0 Hz, 1H), 7.83 (bs, 1H), 7.54 (s, 1H), 7.30-7.25 (m, 2H), 7.11 (t,J=7.6 Hz, 1H), 6.88 (s, 1H), 4.48 (dt, J₁=11.2 Hz, J₂=4.0 Hz, 1H),4.16-4.13 (m, 2H), 3.94 (s, 3H), 3.71 (dd, J₁=16 Hz, J₂=11.2 Hz, 1H),3.47 (d, J=16 Hz, 1H), 2.60 (t, J=6.4 Hz, 2H), 2.22-2.18 (m, 2H).

Compound 127 (IGN-11)

To a flask containing compound 124 (71 mg, 0.099 mmol) was added 4N HClin dioxane (4 mL). The mixture was stirred at room temperature for 2hours and striped under reduced pressure. The residue was dissolved inanhydrous dichloromethane (1.5 mL). Compound 126 (42 mg, 0.11 mmol),triethylamine (14 μl, 0.1 mmol), EDC (38 mg, 0.2 mmol) and DMAP (1 mg,0.0099 mmol) were added subsequently. The reaction mixture was stirredat room temperature for 22 hours and diluted with dichloromethane,washed with saturated ammonium chloride and brine. It was dried overanhydrous sodium sulfate and filtered. The filtrate was striped underreduced pressure and the residue was purified through silica gelchromatography (Combiflash, dichloromethane/MeOH) to furnish compound127 (14 mg, y=49%) as a yellow solid. HRMS (ESI, m/z): calc. 983.4164(M+H)⁺, found 983.4167.

Example 11 Preparation of IGN-03 NHS Ester (Compound 43) and IGN-07 NHSEster (Compound 46) Stock Solution

Solutions of IGN-03 NHS ester and IGN-07 NHS ester are made fresh to a0.006 M stock based on a molecular weight of 903.93 g/mole (IGN-03 NHSester) or 902.95 (IGN-07 NHS ester) in dimethylacetamide (DMA). Thestock solution is assayed spectrophotometrically using a referenceextinction coefficient determined at 330 nm (ε_(330 nm)=15,231 M⁻¹cm⁻¹).

Example 12 4-(tert-Butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxylicacid

Methyl 4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxylate(Eldon E. Baird and Peter B. Dervan, J. Am. Chem. Soc. 1996, 118,6141-6146) (5.0 g, 19.67 mmol) in 120 ml of 1:1 THF/H₂O was added 8 g ofNaOH in 30 ml of water. The mixture was stirred overnight, concentrated,diluted with water, extracted with EtAc/Hexane (1:1). The aqueoussolution was adjusted to pH 4.0 with 20% H₃PO₄ and extracted with EtAc(4×60 ml). The organic solutions were combined, dried over MgSO₄,filtered, evaporated and crystallized with ethanol/EtAc/Hexane to afford3.81 g (81%) of the title product. ¹H NMR (CD₃OD) 12.79 (s, 1H), 10.48(br, 1H), 7.51 (s, 1H), 6.99 (s, 1H), 3.78 (s, 3H), 1.49 (s, 9H); ¹³CNMR 158.47, 153.82, 123.64, 121.56, 109.58, 79.52, 37.06, 28.42; MS m/z−239.2 (M−H).

4-(tert-butoxycarbonylamino)-1-methyl-1H-imidazole-2-carboxylic acid

Methyl 4-(tert-butoxycarbonylamino)-1-methyl-1H-imidazole-2-carboxylate(5.0 g, 19.59 mmol) in 120 ml of 1:1 THF/H₂O was added 8 g of NaOH in 30ml of water. The mixture was stirred overnight, concentrated, dilutedwith water, extracted with EtAc/Hexane (1:1). The aqueous solution wasadjusted to pH 4.0 with 20% H₃PO₄ and extracted with EtAc (4×60 ml). Theorganic solutions were combined, dried over MgSO₄, filtered, evaporatedand crystallized with ethanol/EtAc/Hexane to afford 3.85 g (81%) of thetitle product. ¹H NMR (DMSO) 9.32 (s, 1H), 7.29 (s, 1H), 3.57 (s, 3H),1.42 (s, 9H); ¹³C NMR 172.45, 159.78, 136.93, 135.44, 132.85, 79.50,35.57, 28.07; MS m/z− 240.8 (M−H).

1-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid

Methyl 1-methyl-4-nitro-1H-pyrrole-2-carboxylate (5.0 g, 27.17 mmol) in120 ml of 1:1 THF/H₂O was added 8 g of NaOH in 30 ml of water. Themixture was stirred overnight, concentrated, diluted with water,extracted with EtAc/Hexane (1:1). The aqueous solution was adjusted topH 3˜4 with 20% H₃PO₄ and extracted with EtAc (4×60 ml). The organicsolutions were combined, dried over MgSO₄, filtered, evaporated andcrystallized with ethanol/EtAc/Hexane to afford 4.06 g (88%) of thetitle product. ¹H NMR (DMSO) 13.12 (s, 1H), 8.21 (s, 1H), 7.25 (s, 1H),3.91 (s, 3H); ¹³C NMR 160.97, 134.01, 129.16, 123.81, 111.38, 37.47; MSm/z− 169.1 (M−H).

Methyl1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylate

In a hydrogenation bottle was added methyl1-methyl-4-nitro-1H-pyrrole-2-carboxylate (3.0 g, 16.30 mmol), 80 ml ofTHF, 405 mg of 10% Pd/C and 1.3 ml of HCl (conc.). After evacuationunder vacuum the bottle was placed under 30 psi hydrogen and shaken for5 hours. The mixture was filtered through celites, evaporated to drynesswithout further purification. To the dry mixture was added1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid (2.75 g, 16.18 mmol), 80ml of DMA, EDC (8.51 g, 44.27 mmol) and DIPEA (2.80 ml, 16.10 mmol). Themixture was stirred under Ar overnight, concentrated, diluted withTHF/EtAc (1:2, 150 ml), and washed 1M NaH₂PO₄/NaCl (conc) and NaHCO₃(conc) separately. The organic layer was separated and dried over MgSO₄,filtered concentrated and crystallized with THF/H₂O to afford 3.74 g(75%) of the title product. ¹H NMR (DMSO) 10.25 (s, 1H), 8.17 (s, 1H),7.25 (s, 1H), 6.52 (s, 1H), 6.08 (s, 1H), 3.90 (s, 3H), 3.78 (s, 3H),3.56 (s, 3H); ¹³C NMR 157.87, 156.84, 133.76, 128.16, 123.39, 119.13,118.18, 111.83, 107.50, 104.17, 51.55, 37.41, 36.03; MS m/z+ 329.1(M+Na).

Methyl4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate

Methyl 4-(tert-butoxycarbonylamino)-1-methyl-1H-imidazole-2-carboxylate(2.50 g, 9.80 mmol) in 30 ml of EtAc was added 6 ml of HCl (conc.).After stirring for 45 min, the mixture was diluted with ethanol andtoluene, concentrated and co-evaporated with ethanol/toluene (1:1, 3×50ml) to dryness without further purification. To the dry compound wasadded 4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxylic acid(2.35 g, 9.8 mmol), EDC (5.60 g, 29.1 mmol), DIPEA (1.70 ml, 9.8 mmol)and 80 ml of DMA. The mixture was stirred under Ar overnight,concentrated, diluted with THF/EtAc (1:2, 150 ml), and washed 1MNaH₂PO₄/NaCl (conc) and NaHCO₃ (conc) separately. The organic solventlayer was separated and dried over MgSO₄, filtered, concentrated andpurified on SiO2 chromatography eluted with EtAc/DCM (1:25 to 1:15) toafford 2.72 g (73%) of the title product. ¹H NMR (DMF-d7) 10.27 (s, 1H),9.08 (s, 1H), 7.41 (s, 1H), 7.32 (s, 1H), 7.07 (s, 1H), 4.10 (s, 3H),3.93 (s, 3H), 3.84 (s, 3H), 1.47 (s, 9H); ¹³C NMR 162.62, 161.20,153.82, 145.32, 144.12, 132.56, 128.46, 124.39, 119.83, 79.51, 52.75,36.06, 35.83, 28.88; MS m/z+ 400.2 (M+Na).

Methyl4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate

Methyl 4-(tert-butoxycarbonylamino)-1-methyl-1H-imidazole-2-carboxylate(2.50 g, 9.80 mmol) in 30 ml of EtAc was added 6 ml of HCl (conc.).After stirred for 30 min, the mixture was diluted with ethanol andtoluene, concentrated and co-evaporated with ethanol/toluene (1:1, 3×50ml) to dryness compound without further purification. To the drynesscompound was added4-(tert-butoxycarbonylamino)-1-methyl-1H-imidazole-2-carboxylic acid(2.36 g, 9.8 mmol), EDC (5.90 g, 30.7 mmol), DIPEA (1.70 ml, 9.8 mmol)and 80 ml of DMA. The mixture was stirred under Ar overnight,concentrated, diluted with THF/EtAc (1:2, 150 ml), and washed 1MNaH₂PO₄/NaCl (conc) and NaHCO₃ (conc) separately. The organic solventlayer was separated and dried over MgSO₄, filtered, concentrated andpurified on SiO2 chromatography eluted with EtAc/DCM (1:25 to 1:15) toafford 2.65 g (71.5%) of the title product. ¹H NMR (DMSO) 11.17 (s, 1H),10.48 (s, 1H), 7.58 (s, 1H), 7.32 (s, 1H), 4.01 (s, 3H), 3.94 (s, 3H),3.92 (s, 3H), 1.45 (s, 9H); ¹³C NMR 160.60, 157.30, 135.92, 135.45,132.86, 126.12, 114.83, 79.50, 52.70, 35.58, 34.92, 28.08; MS m/z+ 401.8(M+Na).

1-Methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylicacid

Methyl1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylate(2.0 g, 6.53 mmol) in 50 ml of DMA was added 2 g of LiOH in 30 ml ofwater. The mixture was stirred overnight, concentrated, diluted withwater, extracted with EtAc/Hexane (1:1). The aqueous solution wasadjusted to pH 4.0 with 20% H₃PO₄ to form precipitates. The precipitateswere filtered, washed with water and dried over P₂O₅ with vacuum toafford 1.4 g (73%) of the title product. ¹H NMR (DMF-d7) 10.34 (br, 1H),8.17 (s, 1H), 7.62 (s, 1H), 7.51 (s, 1H), 7.00 (s, 1H), 4.09 (s, 1H),3.91 (s, 1H); ¹³C NMR 158.47, 135.61, 129.11, 127.77, 123.65, 121.57,121.50, 109.48, 108.52, 38.38, 37.05; MS m/z− 291.0 (M−H).

4-(4-(tert-Butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylicacid

Methyl4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylate(2.0 g, 5.30 mmol) in 50 ml of DMA was added 2 g of LiOH in 30 ml ofwater. The mixture was stirred overnight, concentrated, diluted withwater, extracted with EtAc/Hexane (1:1). The aqueous solution wasadjusted to pH 4.0 with 20% H₃PO₄ to form precipitates. The precipitateswere filtered, washed with water and dried over P₂O₅ with vacuum toafford 1.44 g (75%) of the title product. ¹H NMR (DMSO) 10.41 (br, 1H),9.07 (s, 1H), 7.48 (s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 3.92 (s, 1H),3.81 (s, 1H), 1.47 (s, 9H); ¹³C NMR 160.46, 158.42, 152.85, 145.21,135.81, 129.11, 127.77, 122.39, 121.57, 113.58, 79.81, 36.06, 35.25,28.17; MS m/z− 362.1 (M−H).

Methyl4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate

In a hydrogenation bottle was added methyl1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylate(1.0 g, 3.27 mmol), 20 ml of THF, 305 mg of 10% Pd/C (50% wet) and 0.25ml of HCl (conc.). After evacuation under vacuum the bottle was placedunder 50 psi hydrogen and shaken for 4 hours. The mixture was filteredthrough celite, evaporated to dryness without further purification. Tothe dried mixture was added4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxylicacid (1.15 g, 3.16 mmol), 10 ml of DMA, EDC (2.0 g, 10.4 mmol) and DIPEA(0.70 ml, 4.02 mmol). The mixture was stirred under Ar overnight,concentrated, diluted with Hexane/EtAc (1:1, 10 ml) and water 10 ml toform precipitates. The precipitates were filtered, washed 1M NaH₂PO₄, 1M NaHCO₃ and water, dried over P₂O₅ with vacuum to afford 1.61 g (82%)of the title product. ¹H NMR (DMF-d7) 10.29 (s, 1H), 10.20 (s, 1H),10.12 (s, 1H), 9.08 (s, 1H), 7.58 (s, 1H), 7.47 (d, 1H, J=1.7 Hz), 7.26(d, 1H, J=1.5 Hz), 7.15 (d, 1H, J=1.5 Hz), 6.98 (s, 1H), 6.91 (d, 1H,J=1.8 Hz), 6.86 (s, 1H), 3.97 (s, 3H), 3.82 (s, 3H), 3.73 (s, 3H), 3.56(s, 3H), 1.45 (s, 9H); ¹³C NMR 162.16, 160.05, 159.90, 157.20, 154.31,137.88, 135.35, 124.56, 124.39, 124.24, 123.09, 120.09, 119.82, 115.32,105.58, 102.27, 79.31, 51.51, 38.13, 36.01, 35.80, 35.08, 28.79; MS m/z+644.2 (M+Na).

Methyl1-methyl-4-(1-methyl-4-(1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylate

In a hydrogenation bottle was added methyl1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylate(2.0 g, 6.53 mmol), 80 ml of DMA, 500 mg of 10% Pd/C (50% wet) and 0.4ml of HCl (conc.). After evacuation under vacuum, the bottle was placedunder 50 psi hydrogen and shaken for 4 hours. The mixture was filteredthrough celite, evaporated to dryness without further purification. Tothe dry mixture was added1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylicacid (1.49 g, 5.10 mmol), 30 ml of DMA, EDC (4.0 g, 20.8 mmol) and DIPEA(1.0 ml, 5.75 mmol). The mixture was stirred under Ar overnight,concentrated, diluted with Hexane/EtAc (1:1, 10 ml) and water 10 ml toform precipitates. The precipitates were filtered, washed 1M NaH₂PO₄, 1M NaHCO₃ and water, dried over P₂O₅ under vacuum to afford 2.13 g (76%)of the title product. ¹H NMR (DMSO) 10.28 (s, 1H), 10.25 (s, 1H), 9.78(s, 1H), 8.18 (s, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 7.31 (d, 1H, J=1.7Hz), 7.25 (s, 1H), 7.23 (s, 1H), 7.17 (d, 1H, J=1.5 Hz), 6.98 (s, 1H),6.71 (s, 1H), 4.02 (s, 3H), 3.94 (s, 3H), 3.83 (s, 3H), 3.73 (s, 3H),3.56 (s, 3H), 1.47 (s, 9H); ¹³C NMR 160.78, 158.93, 158.06, 157.81,135.25, 127.28, 126.36, 123.78, 122.57, 121.91, 121.40, 120.94, 119.65,110.73, 108.39, 107.34, 103.75, 80.81, 51.57, 39.74, 38.52, 38.22,37.08, 28.63; MS m/z+ 573.2 (M+Na).

4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylicacid

Methyl4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(510 mg, 0.82 mmol) in 10 ml of DMA was added 0.8 g of LiOH in 10 ml ofwater. The mixture was stirred overnight, concentrated, diluted withwater, extracted with EtAc/Hexane (1:1). The aqueous solution wasadjusted to pH 4.0 with 20% H₃PO₄ to form precipitates. The precipitateswere filtered, washed with water and dried over P₂O₅ under vacuum toafford 363 mg (73%) of the title product. ¹H NMR (DMF-d7) 10.31 (s, 1H),10.18 (s, 1H), 10.11 (s, 1H), 9.10 (s, 1H), 7.58 (s, 1H), 7.54 (s, 1H),7.41 (s, 1H), 7.33 (s, 1H), 7.21 (s, 1H), 7.10 (s, 1H), 7.06 (s, 1H),4.10 (s, 1H), 3.98 (s, 1H), 3.95 (s, 1H), 3.93 (s, 1H), 1.47 (s, 9H);¹³C NMR 162.16, 160.05, 159.90, 157.20, 154.31, 137.88, 135.35, 124.56,124.39, 123.51, 123.09, 121.76, 120.09, 119.83, 118.96, 115.32, 109.53,105.58, 102.27, 79.32, 38.13, 36.02, 35.81, 34.88, 28.79; MS m/z− 606.2(M−H).

S-3-(tert-butoxycarbonylamino)propyl ethanethioate

tert-Butyl 3-hydroxypropylcarbamate (3.22 g, 18.37 mmol) in 100 ml ofDCM at 0° C. was added thiolacetic acid (2.0 ml, 26.73 mmol) andtriphenylphosphine (7.0 g, 26.73 mmol) under Ar. After stirred at 0° C.for 15 min, DIAD (6.0 ml, 28.93) was added. The mixture was stirred at0° C. for 2 h then RT overnight. The mixture was concentrated, dilutedwith 120 ml of EtAc/Hexane (1:2), filtered through celite. The solutionwas washed with NaHCO₃ (conc.)/NaCl (conc.) and 1 M NaH₂PO₄respectively, dried over MgSO₄, filtered, evaporated and purified onSiO₂ chromatography eluted with EtAc/Hexane (1:7 to 1:6) to afford 3.22g (75%) of the title compound. ¹H NMR (CDCl₃) 3.09 (t, 2H, J=6.5 Hz),2.84 (t, 2H, J=6.9 Hz), 2.27 (s, 3H), 1.69 (dt, 2H, J=6.8, 13.5 Hz),1.38 (s, 9H); ¹³C NMR 196.35, 156.16, 79.50, 39.26, 30.79, 30.24, 28.61,26.44; MS m/z+ 256.0 (M+Na).

S-3-(tert-butoxycarbonyl(methyl)amino)propyl ethanethioate

To a solution of NaH (0.57 g, 60%, 14.25 mmol) in 20 ml of THF at 0° C.was added S-3-(tert-butoxycarbonylamino)propyl ethanethioate (1.25 g,5.36 mmol) under Ar. After stirring at 0° C. for 30 min, MeI (1.0 ml,16.06 mmol) was added to the mixture. Stirring was continued at 0° C.for 2 h then RT overnight. The mixture was concentrated, redissolved in120 ml of EtAc/Hexane (1:2), washed with 1 M NaH₂PO₄ NaCl (conc.), driedover MgSO₄, filtered, evaporated and purified on SiO₂ chromatographyeluted with EtAc/Hexane (1:7) to afford 1.121 g (85%) of the titlecompound. ¹H NMR (CDCl₃) 3.69 (t, 2H, J=7.3 Hz), 2.41 (t, 2H, J=7.3 Hz),2.39 (s, 3H), 2.03 (s, 3H), 1.76 (m, 2H), 1.47 (s, 9H); ¹³C NMR 173.21,153.39, 83.28, 43.67, 31.84, 28.26, 28.19, 27.11, 15.65; MS m/z+ 270.0(M+Na).

S-3-(Methylamino)propyl ethanethioate hydrogen chloride salt

S-3-(tert-Butoxycarbonyl(methyl)amino)propyl ethanethioate (206 mg,0.834 mmol) in 4 ml of EtAc was added 1.0 ml of HCl (conc.) at RT. Themixture was stirred at RT for 1 h, diluted with ethanol/toluene (6 ml,1:1), evaporated and co-evaporated with ethanol/toluene (3×10 ml),crystallized with ethanol/EtAc/Hexane, filtered, and dried over a vacuumto afford 135 mg (88%) of the title compound. ¹H NMR (CDCl₃) 9.70 (br,1H), 8.56 (br, 1H), 3.42 (m, 2H), 2.52 (m, 2H), 2.35 (s, 3H), 2.05 (s,3H), 1.88 (m, 2H); ¹³C NMR 174.64, 40.57, 31.57, 27.69, 20.94, 15.62; MSm/z+ 170.0 (M+Na), 148.10 (M+H).

tert-Butyl 2-(pyridin-2-yldisulfanyl)ethylcarbamate (217)

To the solution of 2,2′-dithiolpyridine (3.97 g, 18.02 mmol) in 100 mlof methanol and 80 ml of 1 M NaH₂PO₄, pH 6.8 was added tert-Butyl2-mercaptoethylcarbamate (1.00 g, 5.65 mmol) in 50 ml of methanol. Themixture was stirred under Ar overnight, concentrated, extracted withdichloromethane, dried over MgSO₄, filtered, evaporated and purified onSiO₂ chromatography eluted with EtAc/Hexane (1:10 to 1:6) to afford1.341 g (83%) of the title compound. ¹H NMR (CDCl₃) 8.39 (m, 1H), 7.56(m, 1H), 7.49 (m, 1H), 7.03 (m, 1H), 7.00 (m, 1H), 3.34 (m, 2H), 2.84(m, 2H), 1.37 (s, 9H); ¹³C NMR 160.05, 159.39, 159.07, 149.87, 137.21,120.78, 79.48, 39.58, 38.96, 28.57; MS m/z+ 309.2 (M+Na).

2-(pyridin-2-yldisulfanyl)ethanamine

tert-Butyl 2-(pyridin-2-yldisulfanyl)ethylcarbamate (1.06 g, 3.70 mmol)in 16 ml of EtAc was added 4.0 ml of HCl (conc.) at RT. The mixture wasstirred at RT for 0.5 h, diluted with ethanol/toluene (6 ml, 1:1),evaporated and co-evaporated with ethanol/toluene (3×10 ml),crystallized with ethanol/EtAc/Hexane, filtered, and dried over a vacuumto afford 135 mg (88%) of the title compound. ¹H NMR (CD₃OD) 7.58 (m,1H), 7.47 (m, 1H), 7.06 (m, 1H), 6.83 (m, 1H), 3.34 (m, 2H), 3.02 (m,2H); ¹³C NMR 158.69, 149.07, 137.81, 122.48, 120.98, 39.52, 36.94; MSm/z+ 187.10 (M+H).

Methyl 4-bromobutanoate (223)

4-Bromobutanoyl chloride (3.1 ml, 25.28 mmol) was added to 15 ml of drymetanol at 0° C. Stirring was continued at 0° C. under Ar for 2 h thenat RT overnight. The mixture was evaporated, diluted with EtAc/Hexane(1:5), filtered through SiO₂ gel, and evaporated to afford 4.50 g (99%)of the title compound. ¹H NMR (CDCl₃) 3.65 (s, 3H), 3.43 (t, 2H, J=6.5Hz), 2.47 (t, 2H, J=7.1 Hz), 2.13 (dt, 2H, J=6.7, 13.6 Hz); ¹³C NMR173.08, 51.84, 32.82, 32.34, 27.89; MS m/z+ 203.0 (M+Na).

(Z)-methyl4-(7-methoxy-2′,3′-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoate

(Z)-2,3-Benzo-8-hydroxy-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one(60 mg, 0.20 mmol) in 4 ml of acetone was added Cs₂CO₃ (90 mg, 0.28mmol), followed added methyl 4-bromobutanoate (50 mg, 0.27 mmol). Themixture was stirred under Ar over night, evaporated, and purified onSiO₂ chromatography eluted with EtAc/DCM (1:5 to 1:3) to afford 50.1 mg(63%) of the title compound. ¹H NMR (CDCl₃) 8.19 (d, 1H, J=7.9 Hz), 7.80(d, 1H, J=4.2 Hz), 7.48 (s, 1H), 7.19 (m, 2H), 7.03 (d, 1H, J=7.4 Hz),6.77 (s, 1H), 4.41 (m, 1H), 3.88 (s, 3H), 3.64 (m, 2H), 3.62 (s, 3H),3.42 (dd, 1H, J=3.4, 13.7 Hz), 2.50 (t, 2H, J=7.2 Hz), 2.12 (t, 2H,J=6.8 Hz); ¹³C NMR, 173.64, 164.12, 163.24, 152.25, 148.41, 142.28,140.34, 129.69, 128.39, 124.97, 120.85, 117.15, 112.15, 110.68, 68.08,56.40, 55.18, 51.90, 32.84, 30.64, 24.50; MS m/z+ 187.10 (M+H). MS m/z+417.2 (M+Na), 435.2 (M+Na+H₂O).

4-(7-methoxy-2,3-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoic acid

(Z)-methyl4-(7-methoxy-2′,3′-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoate(41 mg, 0.104 mmol) and trimethyltin hydroxide (302 mg, 1.67 mmol) in 15ml of dichloroethane was refluxed at 85° C. under Ar overnight. Themixture was washed with 1 M NaH₂PO₄, pH 3.5, dried over MgSO₄, filtered,evaporated and purified on SiO₂ chromatography eluted with EtAc/DCM/HCl(1:25:0.01%) to afford 30 mg (76%) of the title compound. ¹H NMR (CDCl₃)8.18 (d, 1H, J=7.9 Hz), 7.85 (m, 1H), 7.46 (s, 1H), 7.20 (m, 2H), 7.04(d, 1H, J=7.4 Hz), 6.81 (s, 1H), 4.40 (m, 1H), 3.86 (s, 3H), 3.63 (m,2H), 3.23 (dd, 1H, J=10.2, 16.3 Hz), 2.52 (t, 2H, J=7.2 Hz), 2.12 (t,2H, J=6.8 Hz); ¹³C NMR, 173.64, 164.12, 163.24, 152.25, 148.41, 142.28,140.34, 129.69, 128.39, 125.10, 120.85, 117.19, 112.15, 110.68, 67.94,56.43, 55.18, 31.81, 30.64, 24.21; MS m/z− 397.0 (M+H₂O—H).

4-{[4-({4-[4-(4-(7-methoxy-2′,3′-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butyrylamino]-1-methyl-1H-pyrrole-2-carbonyl}amino)-1-methyl-1H-imidazole-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-carbonyl]-amino}-1-methyl-1H-pyrrole-2-carboxylicacid methyl ester (226)

To methyl4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate(15 mg, 0.024 mmol) in 4 ml of EtAc was added 1.0 ml of HCl (conc.). Themixture was stirred at RT for 0.5 h, diluted with ethanol/toluene (6 ml,1:1), evaporated and co-evaporated with ethanol/toluene (3×10 ml), anddried over a vacuum. The solid compound was used directly withoutfurther purification. To the solid was added4-(7-methoxy-2′,3′-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoic acid (6 mg, 0.015 mmol), EDC (40 mg, 0.21 mmol), DIPEA (4 ul,0.023 mmol) and 1 ml of DMA. The mixture was stirred under Ar overnight, evaporated, and purified on HPLC preparative C-18 column (Φ10mm×200 mm column, flow rate 9 mL/min and a gradient solvent system goingfrom 75:25 solvent A:B at time 0-5 min to 40:60 A:B at 15 min then to20:80 A:B at 25 min until to 10:90 A:B at 30 min. Solvent A—water,solvent B—acetonitrile/dioxane (1:2)) and lyophilized to afford a whitesolid (4.2 mg (30%) of the title compound). MS m/z− 900.3 (M+H₂O—H).

S-3-(4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-N,1-dimethyl-1H-pyrrole-2-carboxamido)propylethanethioate (227)

4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylicacid (256 mg, 0.42 mmol), NHS (60 mg, 0.52 mmol) and EDC (500 mg, 2.60mmol) in 4 ml of DMA were stirred under Ar for 2 h, thenS-3-(methylamino)propyl ethanethioate hydrogen chloride salt (76.5 mg,0.42 mmol) was added and the mixture was kept stirring for 24 h,evaporated and purified on SiO₂ chromatography eluted with THF/DCM (1:5to 1:4) to afford 198 mg (64%) of the title compound. ¹H NMR (DMSO)10.21 (s, 1H), 10.09 (s, 1H), 10.06 (s, 1H), 9.08 (s, 1H), 7.76 (d, 1H,J=1.7 Hz), 7.52 (s, 1H), 7.28 (s, 1H), 7.21 (d, 1H, J=1.7 Hz), 6.97 (s,1H), 6.87 (s, 1H), 3.98 (s, 1H), 3.86 (s, 3H), 3.75 (s, 3H), 3.73 (s,3H), 3.66 (m, 2H), 2.85 (s, 3H), 2.60 (m, 2H), 2.01 (s, 3H), 1.45 (s,9H); ¹³C NMR 173.31, 162.16, 160.05, 159.90, 157.20, 154.31, 137.88,135.35, 124.56, 124.39, 123.51, 123.09, 121.76, 120.09, 119.83, 118.96,115.32, 109.53, 105.58, 102.27, 79.32, 43.67, 38.13, 36.02, 35.81,34.88, 31.84, 28.79, 28.26, 28.21, 27.01; MS m/z+ 759.2 (M+Na).

(Z)—S-3-(4-(4-(4-(4-(4-(7-methoxy-2,3-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-N,1-dimethyl-1H-pyrrole-2-carboxamido)propylethanethioate

S-3-(4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-N,1-dimethyl-1H-pyrrole-2-carboxamido)propylethanethioate (227) (27 mg, 0.037 mmol) was stirred in 2 ml of dioxaneand 0.5 ml of HCl (conc) for 15 min, diluted with ethanol/toluene (6 ml,1:1), evaporated and co-evaporated with ethanol/toluene (4×10 ml),crystallized with EtOH/DCM/Hexane and dried over a vacuum to afford 21mg of solid. The solid compound was used directly without furtherpurification. To the solid was added4-(7-methoxy-2,3-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoic acid (10 mg, 0.026 mmol), EDC (101 mg, 0.52 mmol), DIPEA (5 ul,0.028 mmol) and 2 ml of DMA. The mixture was stirred overnight,evaporated, diluted with DCM, washed with 1 M NaH₂PO₄/NaCl (conc), pH4.0, dried over MgSO₄, filtered, evaporated and purified on HPLCpreparative C-18 column (Φ10 mm×200 mm column, flow rate 9 mL/min and agradient solvent system going from 75:25 solvent A:B at time 0-5 min to40:60 A:B at 15 min then to 20:80 A:B at 25 min until to 10:90 A:B at 30min. Solvent A—water, solvent B—acetonitrile/dioxane (1:2)) andlyophilized to afford a white solid 8.2 mg (32%) of the title compound.MS m/z− 1015.1 (M+H₂O—H), UV ε_((1=305 nm))=32800 M⁻¹cm⁻¹.

tert-Butyl1-methyl-5-(1-methyl-2-(1-methyl-5-(1-methyl-5-(2-(pyridin-2-yldisulfanyl)ethylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-imidazol-4-ylcarbamoyl)-1H-pyrrol-3-ylcarbamate(229)

4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-imidazole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylicacid (102 mg, 0.17 mmol), 2-(pyridin-2-yldisulfanyl)ethanamine hydrogenchloride salt (40 mg, 0.18 mmol), DIPEA (30 ul, 0.17 mmol) and EDC (200mg, 1.04 mmol) in 2 ml of DMA were stirred under Ar for 24 h, evaporatedand purified on SiO₂ chromatography eluted with THF/DCM (1:5 to 1:4) toafford 90 mg (68%) of the title compound. ¹H NMR (DMSO) 10.93 (s, 1H),10.19 (s, 1H), 10.06 (s, 1H), 9.03 (s, 1H), 8.81 (m 1H), 8.29 (m, 1H),8.03 (m, 1H), 7.68 (s, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 7.24 (s, 1H),7.18 (m, 1H), 6.87 (s, 1H), 3.96 (s, 1H), 3.86 (s, 3H), 3.75 (s, 3H),3.73 (s, 3H), 3.58 (m, 2H), 2.48 (m, 2H), 1.45 (s, 9H); MS m/z+798.0(M+Na), 776.0 (M+H).

4-(4-(4-(7-methoxy-2,3-benzo[e]-1-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)-1-methyl-N-(1-methyl-5-(1-methyl-5-(methyl(2-(pyridin-2-yldisulfanyl)ethyl)carbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-yl)-1H-imidazole-2-carboxamide

tert-Butyl1-methyl-5-(1-methyl-2-(1-methyl-5-(1-methyl-5-(2-(pyridin-2-yldisulfanyl)ethylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-imidazol-4-ylcarbamoyl)-1H-pyrrol-3-ylcarbamate(229) (30 mg, 0.038 mmol) was stirred in 2 ml of dioxane and 0.5 ml ofHCl (conc) for 15 min, diluted with ethanol/toluene (6 ml, 1:1),evaporated and co-evaporated with ethanol/toluene (4×10 ml),crystallized with EtOH/DCM/Hexane and dried over vacuum to afford 19.5mg of solid. The solid compound was used directly without furtherpurification. To the solid was added4-(7-methoxy-2,3-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoic acid (10 mg, 0.026 mmol), EDC (102 mg, 0.52 mmol), DIPEA (5 ul,0.028 mmol) and 2 ml of DMA. The mixture was stirred overnight,evaporated, diluted with DCM, washed with 1 M NaH₂PO₄/NaCl (conc), pH4.0, dried over MgSO₄, filtered, evaporated and purified on HPLCpreparative C-18 column (Φ10 mm×200 mm column, flow rate 9 mL/min and agradient solvent system going from 75:25 solvent A:B at time 0-5 min to40:60 A:B at 15 min then to 20:80 A:B at 25 min until to 10:90 A:B at 30min. Solvent A—water, solvent B—acetonitrile/dioxane (1:2)) andlyophilized to afford a white solid 7.5 mg (27%) of the title compound.MS m/z− 1050.0 (M+H₂O—H), UV ε_((1=305 nm))=32855 M⁻¹ cm⁻¹.

1-(4-(2-bromoethoxy)phenyl)ethanone

1-(4-hydroxyphenyl)ethanone (8.2 g, 60.2 mmol), potassium carbonate(15.2 g, 110.1 mmol), and KI (1.0 g, 6.0 mmol) in 100 DMF was stirredfor 5 min, then 1,2-dibromoethane (60 ml, 696.2 mmol) was added. Themixture was stirred overnight, evaporated, diluted with EtAc/Hexane(1:1), washed with 0.1 M HCl/NaCl (conc), dried over MgSO₄, filtered,evaporated and purified by SiO₂ chromatography eluted with EtAc/Hexane(1:3 to 2:3) to afford 12.41 g (85.2%) of the title compound. ¹H NMR(CDCl₃) 7.87 (ddd, 2H, J=2.8, 4.9, 9.7 Hz), 6.88 (ddd, 2H, J=2.8, 4.9,9.6 Hz), 4.29 (t, 2H, J=6.2 Hz), 3.59 (t, 2H, J=6.2 Hz); ¹³C NMR 196.88,162.11, 131.15, 130.54, 113.80, 68.06, 29.50, 26.62; MS m/z+ 264.80(M+Na), 266.80 (M+2+Na).

(5-hydroxy-1,3-phenylene)dimethanol

To a solution of 100 ml of 2.0 M LiAlH₄ in THF at 0° C. was addeddimethyl 5-hydroxy isophthalate (12.3 g, 58.5 mmol) in 120 ml of THF in15 min under Ar. The mixture was stirred at 0° C. for 30 min then at RTovernight. The mixture was quenched with 20 ml of methanol at 0° C., andthe mixture was adjusted to pH 5.0 with addition of H₃PO₄, filteredthrough celite, evaporated and crystallized with ether/hexane to afford76.6 (85%) of the title compound. ¹H NMR (DMSO) 6.68 (s, 1H), 6.61 (s,2H), 4.69 (s, 4H); MS m/z+ 177.0 (M+Na).

1-(4-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)phenyl)ethanone (235)

To a stirred solution of (5-hydroxy-1,3-phenylene)dimethanol (3.0, 20.0mmol), sodium carbonate (2.5 g, 29.0 mmol) and sodium iodide (0.45 g,2.9 mmol) in 60 ml of DMA was added 1-(4-(2-bromoethoxy)phenyl)ethanone(5.0, 20.57 mmol). The mixture was stirred overnight, evaporated andpurified on SiO₂ chromatography eluted with EtAc/Hexane (4:1 to 5:1) toafford 5.41 g (86%) of the title compound. ¹H NMR (CD₃OD) 7.99 (ddd, 2H,J=2.8, 4.8, 9.8 Hz), 7.07 (ddd, 2H, J=2.8, 4.7, 9.8 Hz), 6.94 (s, 1H),6.89 (s, 2H), 4.58 (s, 4H), 4.42 (dd, 2H, J=2.2, 6.1 Hz), 4.37 (m, 2H),2.55 (s, 3H); ¹³C NMR 199.55, 164.66, 160.59, 144.72, 132.03, 131.74,119.16, 115.64, 113.11, 68.36, 67.87, 65.20, 26.53; MS m/z+ 339.2(M+Na).

1-(4-(2-(3,5-bis(bromomethyl)phenoxy)ethoxy)phenyl)ethanone (236)

1-(4-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)phenyl)ethanone (0.216 g,0.68 mmol), carbon tetrabromide (0.50 g, 1.50 mmol) and PPh3 (0.40 g,1.52 mmol) was stirred in 18 ml of THF under Ar overnight and filtered.The solution was concentrated, purified on SiO₂ chromatography elutedwith EtAc/Hexane (1:4) and crystallized with ether/hexane to afford 277mg (92%) of the title compound. ¹H NMR (CDCl₃) 7.94 (ddd, 2H, J=2.7,4.6, 9.6 Hz), 7.02 (s, 1H), 6.98 (ddd, 2H, J=2.7, 4.6, 9.6 Hz), 6.91 (d,2H, J=1.2 Hz), 4.62 (s, 4H), 4.35 (m, 4H), 2.55 (s, 3H); ¹³C NMR 197.05,162.63, 159.14, 139.98, 130.96, 130.85, 122.57, 155.60, 114.52, 66.78,66.73, 32.88, 26.57; MS m/z+ 462.9 (M+Na), 464.9 (M+2+Na).

(R)-Methyl piperidine-2-carboxylate (238)

To (R)-Piperidine-2-carboxylic acid (5.00 g, 38.73) in 150 ml of drymethanol at 0° C. was added thionyl chloride (5.2 ml, 71.28 mmol) underAr. The mixture was stirred at 0° C. for 30 min, then at RT overnight,evaporated and crystallized with EtOH/hexane to afford 4.96 g (92%) ofthe title product. ¹H NMR (CD₃OD) 3.67 (s, 3H), 3.57 (m, 1H), 2.79 (m,1H), 2.69 (m, 1H), 2.01 (m, 1H), 1.98 (m, 1H), 1.73 (m, 1H), 1.55-1.45(m, 4H); ¹³C NMR 171.22, 62.50, 51.35, 45.35, 29.52, 28.41, 23.82; MSm/z+ 144.0 (M+H).

(R)-Methyl1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carboxylate (239)

4-(benzoyloxy)-5-methoxy-2-nitrobenzoic acid (1.70 g, 5.61 mmol),(R)-methyl piperidine-2-carboxylate (1.05 g, 5.84 mmol), EDC (3.90 g,20.31 mmol) and DIPEA (1.0 ml, 5.75 mmol) was stirred in 20 ml of DMAover night. The mixture was evaporated, diluted with DCM, washed withwashed 1M NaH₂PO₄/NaCl (conc) and 0.1 M NaHCO₃/NaCl (conc) separately.The organic solvent layer was separated and dried over MgSO₄, filtered,concentrated and purified on SiO₂ chromatography eluted with EtAc/DCM(1:15) to afford 1.772 g (74%) of the title product. ¹H NMR (CDCl₃) 7.69(s, 1H), 7.40-7.38 (m, 2H), 7.35-7.27 (m, 3H), 6.76 (d, 1H), 5.15 (s,2H), 3.91 (s, 3H), 3.83 (s, 1H), 3.73 (s, 3H), 3.18 (m, 2H), 1.70 (m2H), 1.47 (m, 4H); ¹³C NMR 171.89, 171.33, 155.10, 154.78, 148.32,135.59, 129.05, 128.74, 127.80, 109.66, 109.58, 109.41, 71.63, 56.92,52.70, 52.19, 45.70, 39.92, 27.29, 26.35, 21.63; MS m/z+ 451.2 (M+Na).

(R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carbaldehyde

(R)-Methyl1-(4-(benzoyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carboxylate(1.50 g, 3.50 mmol) in 30 ml of 1:1 DCM/benzene at −78° C. was added 7.5ml of 1.0 M DIBAL in toluene under Ar in 10 min. The mixture was stirredat −78° C. for 1 hr and the reaction was quenched with 0.5 ml ofmethanol. The mixture was diluted with 150 ml of EtAc and 100 ml of 0.2M HCl. The organic solvent layer was separated and was separated and theaqueous layer was extracted with EtOAc (3×80 ml). The organics werecombined, dried over MgSO₄, filtered, concentrated and purified on SiO2chromatography eluted with EtAc/hexane (3:2) to afford 1.52 g (90%) ofthe title product. ¹H NMR (CDCl₃), 9.60 (s, 1H), 7.70 (s, 1H), 7.65-7.28(m, 5H), 6.78 (m, 1H), 5.16 (s, 2H), 3.92 (s, 3H), 3.22, (m, 1H), 3.01(m, 1H), 2.20 (m, 1H), 1.84 (m, 1H), 1.65-1.40 (m, 4H); ¹³C NMR 200.24,171.31, 155.13, 154.78, 148.41, 146.20, 137.57, 135.47, 129.03, 128.73,127.31, 109.83, 109.41, 71.61, 64.50, 56.96, 45.98, 25.25, 23.42, 18.70;MS m/z+ 421.1 (M+Na).

(R,Z)-3-(benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one

To(R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carbaldehyde(1.0 g, 2.51 mmol) in a mixture solution of 25 ml of THF and 15 ml ofwater was added Na₂S₂O₄ (3.0 g, 17.25 mmol). The mixture was stirred for4 h, diluted with methanol and dioxane, evaporated and co-evaporatedwith dioxane (3×60 ml) to dryness. The solid was sonicated with amixture of CH₃OH/CH₂Cl₂ (1:1, 80 ml), filtered and evaporated to solid.The yield solid was dissolved in CH₃OH (100 ml) and 0.4 ml of HCl (conc)was added. The mixture was stirred for 1 h, neutralized to pH 3.0 with0.1 M NaHCO₃, concentrated, and extracted with CH₂Cl₂ (4×60 ml), Theorganic layers were combined, washed with 1M NaHCO₃/NaCl (conc.), driedover Na₂SO₄, filtered, evaporated and purified on SiO₂ chromatographyeluted with EtAc/CH₂Cl₂ (1:3) to afford 615 mg (70%) of the titleproduct. ¹H NMR (CDCl₃), 7.81 (d, 1H, J=5.7 Hz), 7.38˜7.23 (m, 6H), 6.74(s, 1H), 5.12 (dd, 2H, J=2.3, 21.8 Hz), 4.18 (m, 1H), 3.88 (d, 3H), 3.69(m, 1H), 3.15 (m, 1H), 1.99 (m, 1H), 1.87 (m, 1H), 1.79˜1.65 (m, 4H);¹³C NMR 167.76, 163.31, 150.72, 148.48, 140.09, 136.46, 128.87, 128.28,127.53, 121.77, 111.01, 71.02, 56.41, 49.84, 39.93, 24.76, 23.21, 18.62;MS m/z+ 373.2 (M+Na), 391.2 (M+Na+H₂O), 405.3 (M+Na+CH₃OH).

(R,Z)-3-Hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one(242)

To(R,Z)-3-(benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one(241) (215 mg, 0.614 mmol) in 25 ml of CH₂Cl₂ at 0° C. was added 25 mlof CH₂SO₃H. The mixture was stirred at 0° C. for 10 min and then at RTfor 2 h, diluted with CH₂Cl₂, neutralized with cold 1.0 M NaHCO₃,extracted with CH₂Cl₂, dried over Na₂SO₄, filtered, evaporated andpurified on SiO₂ chromatography eluted with CH₃OH/CH₂Cl₂ (1:15) toafford 122 mg (70%) of the title product. ¹H NMR (CDCl₃), 7.75 (d, 1H,J=5.7 Hz), 7.28 (s, 1H), 6.70 (s, 1H), 4.08 (m, 1H), 3.83 (d, 3H), 3.61(m, 1H), 3.08 (m, 1H), 1.91 (m, 1H), 1.81 (m, 1H), 1.71˜1.55 (m, 4H);¹³C NMR 167.81, 163.46, 148.53, 145.71, 140.84, 121.23, 111.89, 111.39,56.45, 49.83, 39.96, 24.71, 23.22, 18.60; MS m/z+ 283.7 (M+Na).

(5Z,5′Z,6aR,6a′R)-3,3′-(5-(2-(4-Acetylphenoxy)ethoxy)-1,3-phenylene)bis(methylene)bis(oxy)bis(2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one)(243)

To a stirring solution of(R,Z)-3-hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one(242) (42 mg, 0.16 mmol), Cs₂CO₃ (100 mg, 0.307 mmol), KI (3.2 mg, 0.018mmol) in 5 ml of acetone was added1-(4-(2-(3,5-bis(bromomethyl)phenoxy)ethoxy)phenyl)ethanone (236) (36mg, 0.081 mmol). The mixture was stirred over night, evaporated andpurified on HPLC preparative C-18 column (Φ10 mm×200 mm column, flowrate 9 mL/min and a gradient solvent system going from 80:20 solvent A:Bat time 0-5 min to 50:50 A:B at 15 min then to 30:70 A:B at 25 min untilto 10:90 A:B at 30 min. Solvent A—water, solvent B—dioxane) andlyophilized to afford a white solid 39.1 mg (61%) of the title compound.¹H NMR (DMF-d₇), 8.30 (m, 2H), 7.75 (d, 2H, J=5.7 Hz), 7.30 (s, 2H),7.01 (m, 2H), 6.71 (s, 2H), 6.68 (s, 1H), 6.63 (s, 2H), 5.21 (s, 4H),4.43 (m, 2H), 4.32 (m, 2H), 4.08 (m, 2H), 3.83 (s, 6H), 3.61 (m, 2H),3.08 (m, 2H), 2.56 (s, 3H), 1.91 (m, 2H), 1.81 (m, 2H), 1.71˜1.55 (m,8H); MS m/z+ 823.2 (M+Na), 839.3 (M+K), 857.3 (M+K+H₂O); MS m/z− 799.2(M−H).

tert-Butyl2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)hydrazinecarboxylate(245)

4-Maleimidobutyric acid (245 mg, 1.33 mmol), tert-butylhydrazinecarboxylate (201 mg, 1.52 mmol) and EDC (400 mg, 2.08 mmol) in5 ml of CH₂Cl₂, were stirred overnight under Ar, washed with 1 MNaH₂PO₄,/NaCl (conc), dried over MgSO₄, filtered, evaporated andpurified on SiO₂ chromatography eluted with MeOH/DCM (1:25) to afford335 mg (85%) of the title compound. ¹H NMR (CDCl₃), 7.83 (br, 1H), 6.65(s, 2H), 6.50 (br, 1H), 3.58 (t, 2H, J=6.3 Hz), 2.15 (t, 2H, J=7.0 Hz),1.90 (dt, 2H, J=6.8, 13.4 Hz), 1.40 (s, 9H); ¹³C NMR 171.30, 155.61,134.41, 82.00, 37.13, 31.38, 28.36, 24.95; MS m/z+ 320.2 (M+Na).

4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanehydrazide trifluoroaceticacid salt (246)

To tert-Butyl2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)-hydrazinecarboxylate(245) (200 mg, 0.673 mmol) in 8 ml of DCM was added 2 ml of TFA. Themixture was stirred for 45 min, diluted with ethanol/toluene (8 ml,1:1), evaporated and co-evaporated with ethanol/toluene (3×10 ml),crystallized with ethanol/EtAc/Hexane, filtered, and dried under vacuumto afford 188 mg (90%) of the title compound. ¹H NMR (CD₃OD) 6.72 (s,2H), 5.39 (s, 0.6H), 3.47 (t, 2H, J=6.6 Hz), 2.20 (m, 2H), 1.85 (m, 2H);¹³C NMR 172.72, 135.56, 54.93, 39.20, 37.99, 25.20; MS m/z+ 197.9 (M+H).

(E)-N′-(1-(4-(2-(3,5-bis(((S,Z)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yloxy)methyl)phenoxy)ethoxy)phenyl)ethylidene)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanehydrazide(247)

4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanehydrazide trifluoroaceticacid salt (246) (3 mg, 0.0096 mmol),(5Z,5′Z,6aR,6a′R)-3,3′-(5-(2-(4-Acetylphenoxy)ethoxy)-1,3-phenylene)bis(methylene)bis(oxy)bis(2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one)(243) (7.5 mg, 0.0093 mmol) and 50 mg 4 Å molecular sieves was stirredin 2 ml of dry 5% HAc in DCM (one day earlier dried by 4 Å molecularsieves) for 2 h, neutralized with 0.5 ml of DIPEA, evaporated andpurified on HPLC preparative C-18 column (Φ10 mm×200 mm column, flowrate 9 mL/min and a gradient solvent system going from 80:20 solvent A:Bat time 0-5 min to 50:50 A:B at 15 min then to 30:70 A:B at 25 min untilto 10:90 A:B at 30 min. Solvent A—water, solvent B—methanol/dioxane(2:1)) and lyophilized to afford a white solid 5.6 mg (61%) of the titlecompound. MS m/z+ 1066.3 (M+2CH₃OH+Na).

Example 13 Preparation of huN901-IGN-07 Conjugate

huN901 antibody that binds to the CD56 antigen was selected forconjugation of IGN derivatives. A solution of huN901 antibody at aconcentration of 3 mg/mL in an aqueous buffer containing 0.05 MN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) and 2 mMethylenediaminetetra-acetic acid (EDTA), pH 8 was treated with a 20-foldmolar excess of a solution of IGN-07 NHS ester in dimethylacetamide(DMA) such that the final concentration of DMA in the buffer was 10%v/v. The reaction mixture was stirred at room temperature for 120 minand then loaded onto a Sephadex G25 gel filtration column (HiPrep™ 26/10Desalting Column GE#17-5087-01) that has been previously equilibratedinto an aqueous buffer containing 0.01 M sodium citrate, 0.135 M sodiumchloride, pH 5.5. The conjugated antibody-containing fractions arecollected and pooled to yield product. The pooled sample was dialyzedovernight against the same elution buffer (0.01 M sodium citrate, 0.135M sodium chloride, pH 5.5) to further purify the product.

The final conjugate was assayed spectrophotometrically using theextinction coefficients that were determined for IGN-07(ε_(330 nm)=15,231 M-1 cm-1 and ε_(280 nm)=26,864 M-1 cm-1) and huN901antibody (ε_(280 nm)=225,000 M-1cm-1). An average of 3.1 IGN moleculesper molecule of antibody were linked.

Preparation of IGN-10 (Compound 51) Stock Solution

A solution of IGN-10 was made fresh to a 0.004 M stock based on amolecular weight of 975.14 g/mole in dimethylacetamide (DMA). The stocksolution was assayed spectrophotometrically using a reference extinctioncoefficient determined at 330 nm (ε_(330 nm)=15,500 M-1 cm-1).

Example 14 Preparation of muB38.1-IGN-10 Conjugate

muB38.1 antibody that binds to the EpCAM antigen was selected forconjugation of IGN derivatives through a disulfide bond. A solution ofmuB38.1 antibody at a concentration of 2.5 mg/mL in an aqueous buffercontaining phosphate buffered saline (PBS) pH 7.4 was treated with 120molar excess of 1-homocysteine thiolactone for 12 hr at 37° C. Thereaction mixture was loaded onto a Sephadex G25 gel filtration column(HiPrep™ 26/10 Desalting Column GE#17-5087-01) that was previouslyequilibrated in PBS pH 7.4. Fractions containing antibody are collectedand pooled and assayed for reactive thiol content using the Ellman'sassay. The modified antibody was then treated with a 4-fold molar excessof IGN-10 (in DMA) per free thiol and allowed to react at roomtemperature for 8 hr. The reaction mixture was loaded onto a SephadexG25 gel filtration column (HiPrep™ 26/10 Desalting Column GE#17-5087-01)that has been previously equilibrated into an aqueous buffer containing0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5. The conjugatedantibody-containing fractions are collected and pooled to yield product.The pooled sample was dialyzed overnight against the same elution buffer(0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5) to furtherpurify the product.

The final conjugate was assayed spectrophotometrically using theextinction coefficients that were determined for IGN-10(ε_(330 nm)=15,500 M-1 cm⁻¹ and ε_(280 nm)=26,864 M-1 cm⁻¹) and muB38.1antibody (ε_(280 nm)=215,525 M-1cm-1). An average of 0.7 IGN moleculesper molecule of antibody was linked.

Example 15 DNA Probe Assay for Measuring IGN Dimer Binding andAlkylation to Double Stranded DNA (dsDNA)

Reaction conditions: dsDNA (25 μM final concentration) in 100 mM TRIS, 1mM EDTA, pH 8 was mixed with 3.7 molar equivalents of IGN-01 (compound18), IGN-02 (compound 19), or IGN-09 (compound 15) dissolved inacetonitrile (final acetonitrile concentration <2% by volume). Thereaction was incubated at 15° C. (below TM of the dsDNA) and 10 μlaliquots are injected on reverse phase-HPLC at various time points aftermixing

HPLC conditions: Waters Xbridge C8 2.1×50 mm column, Buffer A: 100 mMhexafluoroisopropanol, 16.3 mM triethylamine, in water, Buffer B:Methanol; 98% A→100% B over 32 min, 0.25 ml/min flow, 60° C. columnheat, 260 nm detection. Areas under the curve (AUC) for the probe DNApeak and the resulting IGN/DNA adduct peak are used to determine the %crosslinking at each time point of incubation.

DNA annealing: single stranded DNA (Invitrogen) was annealed into dsDNAusing a Peltier thermal cycler (PTC-200, MJ Research). 1 mM DNA in 100mM TRIS, 1 mM EDTA pH 8 was heated to 80° C. and then gradually cooledto 4° C. over 90 min in 15 degree steps. The resulting dsDNA was kept at4° C. until used in the assay. IGN-01, IGN-02, and IGN-09 did not formcovalent adducts with single stranded DNA (ssDNA) in controlexperiments.

Example 16

2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

To a stirred solution of 2-(2-(2-methoxyethoxy)ethoxy)ethanol (1.64 g,10 mmol) in anhydrous dichloromethane (30 mL) was added triethylamine(2.53 g, 25 mmol), tosyl chloride (3.81 g, 20 mmol) and DMAP (0.061 g,0.5 mmol) subsequently at room temperature. The mixture continued to bestirred overnight and worked up by diluted with ethyl acetate andfiltered to remove the triethylamine hydrochloride solid. The solid waswashed with ethyl acetate and the filtrate was evaporated. The residuewas diluted with ethyl acetate and filtered to remove the additionalprecipitate. The filtrate was evaporated to give the crude product asliquid. It was purified by silica gel chromatography(dichloromethane/methanol) to give compound 249a as an oil (3.16 g,yield=99%). ¹H NMR (400 Hz, CDCl₃): δ 7.81 (d, J=8.0 Hz, 2H), 7.35 (d,J=8.0 Hz, 2H), 4.17 (t, J=3.2 Hz, 2H), 3.70 (t, J=4.8 Hz, 2H), 3.64-3.60(m, 6H), 3.54 (t, J=4.8 Hz, 2H), 3.38 (s, 3H), 2.46 (s, 3H); ¹³C NMR(400 Hz, CDCl₃): δ 144.7, 133.0, 129.8, 127.9, 71.9, 70.7, 70.52, 70.50,69.2, 68.6, 59.0, 21.6; MS (m/z): found 341.1 (M+Na)⁺.

(5-(2-(2-(2-methoxyethoxy)ethoxy)ethylamino)-1,3-phenylene)dimethanol

To the mixture of the tosylate 249a (1.85 g, 5.81 mmol) and anilinecompound 26 (1.78 g, 11.6 mmol) in anhydrous DMF (9 mL) was addedanhydrous potassium carbonate (1.61 g, 11.6 mmol). The mixture washeated to 85° C. and stirred at that temperature overnight. The solutionwas cooled to room temperature and diluted with dichloromethane. It wasfiltered through celite and the solid was washed with dichloromethane.The filtrate was evaporated and the residue was diluted withdichloromethane and filtered again to remove the additional solid. Thefiltrate was evaporated and the residue was purified by silica gelchromatography (dichloromethane/methanol) to give compound 249b as alight yellowish oil (835 mg, yield=48%). ¹H NMR (400 Hz, CDCl₃): δ 6.60(s, 1H), 6.47 (s, 2H), 4.48 (s, 4H), 4.31 (bs, 1H), 3.66-3.59 (m, 8H),3.55-3.52 (m, 2H), 3.36 (s, 3H), 3.24 (t, J=4.8 Hz, 2H); ¹³C NMR (400Hz, CDCl₃): δ 148.5, 142.4, 114.6, 110.7, 71.8, 70.4, 70.3, 70.1, 69.4,64.9, 58.9, 43.5; MS (m/z): found 322.2 (M+Na)⁺.

Compound 249c (IGN-14 Linker)

To the solution of compound 249b (319 mg, 1.07 mmol) and methyl4-bromobutyrate (248 mg, 1.37 mmol) in anhydrous acetonitrile (5 mL) wasadded anhydrous potassium carbonate (177 mg, 1.28 mmol). The mixture wasstirred and heated at reflux (86° C. oil bath) overnight. It was cooledto room temperature and diluted with dichloromethane. The mixture wasfiltered through celite and the filtrate was evaporated. The residue waspurified by silica gel chromatography (dichloromethane/methanol) to givecompound 249c (IGN-14 linker) as colorless oil (246 mg, yield=58%). ¹HNMR (400 Hz, CDCl₃): δ 6.69 (s, 2H), 6.66 (s, 1H), 4.64 (s, 4H), 3.71(s, 3H), 3.64-3.62 (m, 8H), 3.57-3.54 (m, 4H), 3.40-3.38 (m, 5H), 2.38(t, J=7.2 Hz, 2H), 1.93 (p, J=7.2 Hz, 2H); MS (m/z): found 422.3(M+Na)⁺.

Compound 249d (IGN-14-OMe)

To a stirred solution of compound 249c (120 mg, 0.3 mmol) in anhydrousdichloromethane (3 mL) was added triethylamine (146 μl, 1.05 mmol). Themixture was cooled to −10° C. and methanesulfonyl chloride (70 μl, 0.9mmol) was added slowly in 15 minutes. The solution continued to bestirred between −10° C. to −5° C. for 60 minutes and quenched byaddition of ice/water. It was diluted with ethyl acetate and washed withcold water. The organic layer was dried over anhydrous sodium sulfate,filtered, evaporated and high vacuumed to give the mesylate as colorlessoil. The mesylate was transferred to a 10 mL round bottom flask withethyl acetate, evaporated and high vacuumed. Compound 8 (221 mg, 0.75mmol) was added followed by addition of anhydrous DMF (2 mL) andanhydrous potassium carbonate (207 mg, 1.5 mmol). The mixture wasstirred at room temperature overnight. It was diluted withdichloromethane and washed with brine. The organic layer was dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by preparative reverse phase HPLC (C18 column, eluted withCH₃CN/H₂O) to give compound 249d (IGN-14-OMe) as a light yellowish solid(98 mg, yield=34%). ¹H NMR (400 Hz, CDCl₃): δ 8.29 (d, J=8.0 Hz, 2H),7.86 (d, J=4.4 Hz, 2H), 7.58 (s, 2H), 7.31-7.26 (m, 4H), 7.12 (t, J=8.0Hz, 2H), 6.88 (s, 2H), 6.83 (s, 1H), 6.76 (s, 2H), 5.18 (dd, J₁=23.2 Hz,J₂=12.4 Hz, 4H), 4.49 (dt, J₁=10.8 Hz, J₂=4.4 Hz, 2H), 3.99 (s, 6H),3.73-3.52 (m, 19H), 3.40-3.37 (m, 5H), 2.35 (t, J=7.2 Hz, 2H), 1.90 (p,J=7.2 Hz, 2H); ¹³C NMR (400 Hz, CDCl₃): δ 173.7, 164.9, 163.2, 151.1,148.5, 148.4, 142.1, 140.2, 137.8, 129.7, 128.2, 124.9, 120.7, 117.0,113.8, 112.0, 111.4, 110.4, 72.0, 71.3, 70.7, 70.6, 68.6, 59.1, 56.3,55.0, 51.7, 50.7, 32.7, 31.3, 22.4; MS (m/z): found 974.6 (M+Na)⁺, 992.7(M+H₂O+Na)⁺, 1010.7 (M+2H₂O+Na)⁺, 950.3 (M−H)⁻, 1022.3 (M+4H₂O—H)⁻.

Compound 249f (IGN-14-NHS)

To the solution of compound 249d (105 mg, 0.11 mmol) in anhydrous 1,2dichloroethane (2 mL) was added trimethyltin hydroxide (299 mg, 1.65mmol). The mixture was heated to 80° C. and stirred overnight. It wascooled to room temperature, diluted with dichloromethane and washed withmixed solution of saturated sodium chloride and 5% hydrochloric acid (˜1mL), then brine. The organic layer was dried over anhydrous sodiumsulfate, filtered and evaporated. The residue was passed a short silicagel column and eluted with dichloromethane/methanol to remove the extratrimethyltin hydroxide. The product fractions were evaporated and highvacuumed to give the acid 249e as a yellowish solid (102 mg, yield=99%).MS (m/z): found 936.1 (M−H)⁻, 960.3 (M+Na)⁺. Compound 249e was thendissolved in anhydrous dichloromethane (1 mL). N-hydroxysuccinimide(NHS, 37.5 mg, 0.326 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 62.5mg, 0.326 mmol) was added subsequently. The mixture was stirred at roomtemperature overnight and diluted with dichloromethane, washed withbrine and dried over anhydrous sodium sulfate. It was filtered,evaporated and the residue was purified by preparative reverse phaseHPLC (C18 column, eluted with acetonitrile/water). The product fractionswere combined and extracted with dichloromethane. The organic layerswere dried over anhydrous sodium sulfate, filtered, evaporated and highvacuumed to give compound 249f (IGN-14-NHS) as a light yellowish solid(57.8 mg, yield=51%). ¹H NMR (400 Hz, CDCl₃): δ 8.28 (d, J=7.6 Hz, 2H),7.86 (d, J=4.4 Hz, 2H), 7.58 (s, 2H), 7.31-7.27 (m, 4H), 7.12 (t, J=7.6Hz, 2H), 6.87 (s, 2H), 6.81 (s, 1H), 6.74 (s, 2H), 5.23 (dd, J₁=26.4 Hz,J₂=12.4 Hz, 4H), 4.49 (dt, J₁=10.8 Hz, J₂=4.4 Hz, 2H), 4.00 (s, 6H),3.73-3.47 (m, 18H), 3.37 (s, 3H), 2.79-2.74 (m, 4H), 2.59 (t, J=7.2 Hz,2H), 1.97 (p, J=7.2 Hz, 2H); MS (m/z): found 1057.4 (M+Na)⁺.

Example 17

Compounds 250a (IGN-16-OMe) and 250b (IGN-18-OMe)

To a stirred solution of compound 34 (111 mg, 0.135 mmol) in absoluteethanol (1.0 mL) and anhydrous dichloromethane (0.5 mL) was added sodiumborohydride (1.0 mg, 0.027 mmol) at 0° C. After 30 minutes, theice/water bath was removed and the reaction mixture continued to bestirred at room temperature for 3 hours. The reaction was quenched byaddition of saturated ammonium chloride and diluted withdichloromethane. The mixture was separated and the organic layer waswashed with brine, dried over anhydrous sodium sulfate and filtered. Thesolvents were removed under reduced pressure and the residue waspurified by preparative reverse phase HPLC (C18 column, eluted withacetonitrile/water) to give compounds 250a (IGN-16-OMe, 6.6 mg) and 250b(8.0 mg) as white solid. 250a: MS (m/z): found 845.3 (M+Na)⁺, 863.3(M+H₂O+Na)⁺. 250b: ¹H NMR (400 Hz, CDCl₃), δ 8.34 (d, J=8.0 Hz, 2H),7.49 (s, 2H), 7.27-7.03 (m, 6H), 6.89-6.87 (m, 3H), 6.05 (s, 2H), 4.96(dd, J₁=20.8 Hz, J₂=12.8 Hz, 4H), 4.40-4.34 (m, 2H), 3.94-3.91 (m, 2H),3.87 (s, 6H), 3.67 (s, 3H), 3.53-3.42 (m, 6H), 2.78 (dd, J₁=16.8 Hz,J₂=4.0 Hz, 2H), 2.38-2.37 (m, 2H), 1.79-1.77 (m, 4H); MS (m/z): found847.3 (M+Na)⁺.

Example 18

Compound 251a

To a stirred solution of compound 5 (840 mg, 1.82 mmol) in ethyl acetate(10 mL) was added palladium on charcoal (10%, 193 mg, 0.182 mmol). Theflask was briefly vacuumed and replaced with H₂ in a balloon. Themixture continued to be hydrogenated for overnight and filtered throughcelite. The solid was washed with methanol and the filtrate was treatedwith 5% hydrochloric acid (0.1 mL). The solution was stripped underreduce pressure and the residue was purified by silica gelchromatography (dichloromethane/methanol) to give compound 251a as awhite solid (512 mg, yield=91%). ¹H NMR (400 Hz, CDCl₃), δ 8.21 (d,J=8.0 Hz, 1H), 8.09 (bs, NH), 7.53 (s, 1H), 7.31-7.25 (m, 2H), 7.12 (t,J=7.6 Hz, 1H), 6.61 (s, 1H), 6.22 (bs, 1H), 4.73 (dd, J₁=10.4 Hz, J₂=2.8Hz, 1H), 4.07 (dd, J₁=16.4 Hz, J₂=2.4 Hz, 1H), 3.98 (s, 3H), 3.34 (dd,J₁=16.4 Hz, J₂=10.4 Hz, 1H); MS (m/z): found 333.1 (M+Na)⁺, 308.9 (M−H)⁻

Compound 251b (IGN-17-OMe)

To a solution of compound 38 (0.165 mmol, prepared from 44 mg ofcompound 30 following the procedure described in example 6) and 251a(128 mg, 0.413 mmol) in anhydrous DMF (1.5 mL) was added anhydrouspotassium carbonate (114 mg, 0.825 mmol). The mixture was stirred atroom temperature overnight and diluted with dichloromethane, washed withbrine and dried over anhydrous sodium sulfate and magnesium sulfate. Itwas filtered, evaporated and part of the residue was purified bypreparative reverse phase HPLC (C18 column, eluted withacetonitrile/water) to give 1.9 mg of compound 251b as a white solid.The rest of the residue was purified by preparative thin layerchromatography (dichloromethane/methanol, 12:1) to give 36.8 mg ofproduct as a white solid. Total 38.7 mg of compound 251b (IGN-17-OMe)was isolated (yield=28%). ¹H NMR (400 Hz, CDCl₃): δ 8.61 (s, 2H), 8.15(d, J=8.0 Hz, 2H), 7.48 (s, 2H), 7.25 (d, J=7.6 Hz, 2H), 7.20 (t, J=7.6Hz, 2H), 7.07 (t, J=7.6 Hz, 2H), 6.73 (s, 1H), 6.69 (s, 2H), 6.58 (s,2H), 5.02 (dd, J₁=17.6 Hz, J₂=13.2 Hz, 4H), 4.66 (dd, J₁=10.4 Hz, J₂=2.8Hz, 2H), 4.00 (dd, J₁=16.4 Hz, J₂=2.4 Hz, 2H), 3.90 (s, 6H), 3.68 (s,3H), 3.39-3.23 (m, 4H), 2.89 (s, 3H), 2.44-2.30 (m, 2H), 1.91-1.92 (m,2H); ¹³C NMR (400 Hz, CDCl₃): δ 174.5, 169.1, 164.2, 151.6, 149.6,146.9, 141.2, 137.3, 130.6, 129.4, 127.5, 124.9, 124.8, 119.6, 117.1,114.2, 112.5, 110.9, 106.0, 71.4, 58.0, 56.2, 51.9, 51.7, 38.3, 31.1,28.2, 21.8; MS (m/z): found 874.3 (M+Na)⁺, 850.2 (M−H)⁻.

Example 19

Compound 252a

The mixture of 1,3-dibromoaceton (0.863 g, purity 75%, 3.0 mmol) andmethyl(triphenylphosphoranylidene)acetate (1.505 g, 4.5 mmol) inanhydrous THF (15 mL) was heated to reflux for 4.5 hours. The solutionwas cooled to room temperature and evaporated. The residue was purifiedby silica gel chromatography (hexanes/ethyl acetate) to give compound252a as colorless liquid (485 mg, yield=60%). ¹H NMR (400 Hz, CDCl₃): δ6.06 (s, 1H), 4.76 (s, 2H), 4.19 (s, 2H), 3.79 (s, 3H); ¹³C NMR (400 Hz,CDCl₃): δ 165.1, 150.4, 121.3, 51.8, 33.6, 25.5.

Compound 252b (IGN-19-OMe)

The mixture of compound 252a (32 mg, 0.118 mmol), monomer 8 (87 mg,0.294 mmol) and anhydrous potassium carbonate (49 mg, 0.353 mmol) inanhydrous DMF (1 mL) was stirred at room temperature overnight. It wasdiluted with dichloromethane, washed with brine and dried over anhydroussodium sulfate. The solution was filtered, evaporated and purified bysilica gel chromatography (dichloromethane/methanol) to give 105 mg ofcompound 252b mixed with side products as yellowish foam. Part of theproducts was further purified by preparative reverse phase HPLC (C18column, eluted with acetonitrile/water) to give 10 mg of compound 252b(IGN-19-OMe) as a white solid. MS (m/z): found 721.2 (M+Na)⁺, 739.2(M+H₂O+Na)⁺, 757.2 (M+2H₂O+Na)⁺, 697.1 (M−H)⁻, 769.1 (M+4H₂O—H)⁻.

Example 20

Compound 253a

To a solution of 2-(methyldithio)-isobutyraldehyde (690 mg, 4.59 mmol)in absolute ethanol (15 mL) was added methylamine (629 μl, 33% wt, 5.05mmol). The mixture was stirred at room temperature for four hours andcooled to 0° C. followed by addition of sodium borohydride (174 mg, 4.59mmol). After one hour, the reaction was quenched with a few drops ofcold 5% hydrochloric acid and then basified with saturated sodiumbicarbonate. The mixture was diluted with dichloromethane and washedwith brine. The organic layer was dried over anhydrous sodium sulfate,filtered and evaporated under reduce pressure. The residue was purifiedby silica gel chromatography (0.2% triethylamine indichloromethane/methanol) to give volatile compound 253a as lightyellowish liquid (491 mg, yield=65%). ¹H NMR (400 Hz, CDCl₃): δ 2.61 (s,2H), 2.45 (s, 3H), 2.39 (s, 3H), 1.32 (s, 6H), 1.20 (s, NH); ¹³C NMR(400 Hz, CDCl₃): δ 61.2, 51.7, 37.2, 26.5, 25.3; MS (m/z): found 166.0(M+H)⁺.

Compound 253b

The mixture of compound 249c (117 mg, 0.293 mmol) and trimethyltinhydroxide (794 mg, 4.39 mmol) in anhydrous 1,2-dichloroethane (1.5 mL)was heated to 80° C. and stirred overnight. It was cooled to roomtemperature, diluted with dichloromethane and washed with mixed solutionof saturated sodium chloride and 5% hydrochloric acid (˜1 mL), thenbrine. The organic layer was dried over anhydrous sodium sulfate,filtered and evaporated. The residue was purified by silica gelchromatography (dichloromethane/methanol) to give the acid 253b as acolorless oil (93.9 mg, yield=99%). ¹H NMR (400 Hz, CDCl₃): δ 6.62 (s,2H), 6.57 (s, 1H), 4.50 (s, 4H), 3.63-3.54 (m, 8H), 3.53-3.46 (m, 4H),3.36-3.31 (m, 5H), 2.29 (t, J=6.8 Hz, 2H), 1.83 (p, J=6.8 Hz, 2H); ¹³CNMR (400 Hz, CDCl₃): δ 177.0, 148.2, 142.4, 113.8, 110.1, 71.9, 70.7,70.6, 70.4, 68.8, 65.2, 59.0, 50.8, 50.7, 31.4, 22.3; MS (m/z): found384.2 (M−H)⁻, 408.4 (M+Na)⁺.

Compound 253c

To a solution of amine 253a (44.3 mg, 0.268 mmol) and carboxylic acid253b (93.3, 0.244 mmol) in anhydrous dichloromethane (1.5 mL) was addedN-hydroxysuccinimide (NHS, 70.1 mg, 0.365 mmol) and DMAP (5.95 mg, 0.049mmol). The mixture was stirred at room temperature overnight and dilutedwith dichloromethane, washed with saturated ammonium chloride and brine,dried over anhydrous sodium sulfate, filtered and evaporated. Theresidue was purified by silica gel chromatography(dichloromethane/methanol) to give compound 253c as colorless oil (69.1mg, yield=53%). ¹H NMR (400 Hz, CDCl₃): δ 6.71 (s, 2H), 6.64 (s, 1H),4.57 (s, 4H), 3.63-3.59 (m, 8H+2OH), 3.54-3.51 (m, 4H), 3.38-3.34 (m,5H), 3.08 (s, 2.35H), 3.00 (s, 0.65H), 2.86 (bs, 2H), 2.43 (s, 3H), 2.34(t, J=6.8 Hz, 2H), 1.95-1.88 (m, 2H), 1.36 (s, 1.3H), 1.31 (s, 4.7H);¹³C NMR (400 Hz, CDCl₃): δ 173.7, 148.5, 142.7, 113.2, 109.8, 72.0,70.8, 70.7, 70.6, 68.9, 65.6, 59.1, 56.5, 53.0, 52.2, 51.0, 50.8, 38.8,30.6, 26.6, 25.6, 22.3; MS (m/z): found 555.5 (M+Na)⁺.

Compound 253d

To a stirred solution of compound 253c (69.1 mg, 0.13 mmol) in anhydrousdichloromethane (1.5 mL) was added triethylamine (63 μl, 0.454 mmol).The mixture was cooled to −10° C. and methanesulfonyl chloride (30 μl,0.389 mmol) was added slowly in 15 minutes. The solution continued to bestirred between −10° C. to −5° C. for 60 minutes and quenched byaddition of ice/water. It was diluted with ethyl acetate and washed withcold water. The organic layer was dried over anhydrous sodium sulfate,filtered, evaporated and high vacuumed to give the mesylate as colorlessoil. The mesylate was transferred to a 10 mL round bottom flask withethyl acetate, evaporated and high vacuumed. Compound 8 (99 mg, 0.338mmol) was added followed by addition of anhydrous DMF (1 mL) andanhydrous potassium carbonate (90 mg, 0.65 mmol). The mixture wasstirred at room temperature overnight. It was diluted withdichloromethane and washed with brine. The organic layer was dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel chromatography (dichloromethane/methanol) to give150 mg yellowish foam, which was further purified by preparative reversephase HPLC (C18 column, eluted with CH₃CN/H₂O) to give compound 253d asa light yellowish solid (50.7 mg, yield=36%). MS (m/z): found 1107.7(M+Na)⁺, 1125.7 (M+H₂O+Na)⁺, 1143.7 (M+2H₂O+Na)⁺, 1083.4 (M−H)⁻, 1155.5(M+4H₂O—H)⁻.

Compound 253e

To a small vial containing tris(2-carboxyethyl)phosphine hydrochloride(TCEP, 19.1 mg, 0.067 mmol) was added a few drops of deioned water.Saturated sodium bicarbonate was added dropwise until pH is about 7indicated by a pH test paper. It was then diluted with pH 6.5 phosphatebuffer (0.4 mL) to give a fresh TCEP solution. To a stirred solution ofcompound 253d (24.1 mg, 0.022 mmol) in methanol (3 mL) and acetonitrile(1 mL) was added the TCEP solution and stirred at room temperature for1.5 hours. It was diluted with dichloromethane and washed with brine,dried over anhydrous sodium sulfate, filtered and evaporated to give thethiol as a yellowish solid (21.9 mg) which was directly used for nextstep (the thiol is not able to be purified due to aggregation). To asolution of the thiol (21.9 mg, 0.021 mmol) in anhydrous dichloromethane(0.1 mL) and methanol (0.4 mL) was added 4-(2-pyridyldithio)butanoicacid (24.2 mg, 0.105 mmol) and triethyl amine (29 μl, 0.211 mmol). Themixture was stirred at room temperature for five hours and quenched bysaturated ammonium chloride. It was diluted with dichloromethane,separated and the organic layer was washed with brine, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by preparative reverse phase HPLC (C18 column, eluted withacetonitrile/water) to give compound 253e as a white solid (7.3 mg,yield=30%). ¹H NMR (400 Hz, CDCl₃): δ 8.28 (d, J=7.6 Hz, 2H), 7.89 (bs,2H), 7.60 (bs, 2H), 7.31-7.26 (m, 4H), 7.12 (t, J=7.6 Hz, 2H), 6.91-6.78(m, 5H), 5.22-5.13 (m, 4H), 4.54-4.49 (m, 2H), 3.99 (s, 6H), 3.68-3.41(m, 20H), 3.38 (s, 3H), 3.07 (s, 3H), 2.78-2.77 (m, 2H), 2.47 (bs, 2H),2.35 (bs, 2H), 2.01-1.95 (m, 4H), 1.31 (s, 6H); MS (m/z): found 1179.7(M+Na)⁺, 1197.7 (M+H₂O+Na)⁺, 1073.6 (M+H₂O—H)⁻, 1191.5 (M+2H₂O—H)⁻.

Compound 253f

To a solution of compound 253e (9.0 mg, 0.00778 mmol) in anhydrousdichloromethane (0.5 mL) was added N-hydroxysuccinimide (NHS, 2.68 mg,0.023 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC, 4.47 mg, 0.023 mmol) subsequently. The mixture wasstirred at room temperature overnight and diluted with dichloromethane,washed with brine and dried over anhydrous sodium sulfate. It wasfiltered, evaporated and the residue was purified by preparative reversephase HPLC (C18 column, eluted with acetonitrile/water). The productfractions were combined and extracted with dichloromethane. The organiclayers were dried over anhydrous sodium sulfate, filtered, evaporatedand high vacuumed to give compound 253f (IGN-20-NHS) as a yellowish foam(4.4 mg, yield=45%). MS (m/z): found 1276.7 (M+Na)⁺.

Example 21

Compound 254a (IGN-23-OMe) and 254b (IGN-24-OMe)

To a stirred solution of compound 249d (91.8 mg, 0.103 mmol) in absoluteethanol (1.0 mL) and anhydrous dichloromethane (0.4 mL) was added sodiumborohydride (0.4 mg, 0.0106 mmol) at 0° C. After 30 minutes, theice/water bath was removed and the reaction mixture continued to bestirred at room temperature for 3 hours. The reaction was quenched byaddition of saturated sodium ammonium chloride and diluted withdichloromethane. The mixture was separated and the organic layer waswashed with brine, dried over anhydrous sodium sulfate and filtered. Thesolvents were removed under reduced pressure and the residue waspurified by preparative reverse phase HPLC (C18 column, eluted withacetonitrile/water) to give compounds 254a (IGN-23-OMe, 24.2 mg,yield=24.5%) and 254b (IGN-24-OMe, 26.3 mg, yield=26.6%) as a yellowishsolid. 254a: ¹H NMR (400 Hz, CDCl₃): δ 8.34 (d, J=8.0 Hz, 1H), 8.27 (d,J=7.6 Hz, 1H), 7.83 (d, J=4.4 Hz, 1H), 7.57 (s, 1H), 7.46 (s, 1H),7.29-7.02 (m, 6H), 6.87 (s, 1H), 6.75 (s, 1H), 6.70-6.66 (m, 2H), 6.10(s, 1H), 5.21-5.02 (m, 4H), 4.49-4.39 (m, 2H), 3.99 (s, 3H), 3.89 (s,3H), 3.66 (s, 3H), 3.64-3.41 (m, 19H), 3.39-3.34 (m, 4H), 2.78 (dd,J₁=16.4 Hz, J₂=3.6 Hz, 1H), 2.33 (t, J=7.2 Hz, 2H), 1.90-1.84 (m, 2H);¹³C NMR (400 Hz, CDCl₃): δ 173.8, 166.8, 164.0, 163.5, 152.3, 151.2,148.7, 148.5, 143.0, 142.1, 140.7, 140.2, 138.5, 137.8, 129.8, 129.7,128.3, 127.9, 125.0, 124.7, 123.9, 120.9, 117.5, 117.0, 114.6, 113.4,113.2, 112.1, 111.6, 110.2, 110.1, 104.2, 72.1, 71.4, 71.2, 70.80,70.76, 70.70, 68.7, 59.2, 57.3, 56.5, 56.4, 55.1, 54.8, 51.8, 50.9,50.7, 33.3, 32.7, 31.3, 22.4; MS (m/z): found 976.7 (M+Na)⁺, 994.6(M+H₂O+Na)⁺; 254b: MS (m/z): found 978.7 (M+Na)⁺.

Compound 254c and 254d (IGN-23-NHS)

To the solution of compound 254a (31.8 mg, 0.033 mmol) in anhydrous 1,2dichloroethane (1 mL) was added trimethyltin hydroxide (90 mg, 0.5mmol). The mixture was heated to 80° C. and stirred overnight. It wascooled to room temperature, diluted with dichloromethane and washed withmixed solution of saturated sodium chloride and 5% hydrochloric acid (˜1mL), then brine. The organic layer was dried over anhydrous sodiumsulfate, filtered and evaporated. The residue was passed a short silicagel column and eluted with dichloromethane/methanol to remove the extratrimethyltin hydroxide. The product fractions were evaporated and highvacuumed to give the acid 254c as a yellowish solid (20.8 mg,yield=66%). MS (m/z): found 938.2 (M−H)⁻, 962.3 (M+Na)⁺. Compound 254c(20.8 mg, 0.022 mmol) was then dissolved in anhydrous dichloromethane (1mL). The reaction flask was briefly vacuumed and replaced with argon.N-hydroxysuccinimide (NHS, 5.09 mg, 0.044 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 8.48mg, 0.044 mmol) was added subsequently. The mixture was stirred at roomtemperature overnight and diluted with dichloromethane, washed withbrine and dried over anhydrous sodium sulfate. It was filtered,evaporated and the residue was purified by preparative reverse phaseHPLC (C18 column, eluted with acetonitrile/water). The product fractionswere combined and extracted with dichloromethane. The organic layerswere dried over anhydrous sodium sulfate, filtered, evaporated and highvacuumed to give compound 254d (IGN-23-NHS) as a light yellowish solid(9.8 mg, yield=43%). MS (m/z): found 1059.6 (M+Na)⁺, 1077.6 (M+H₂O+Na)⁺.

Compound 254e and 254f (IGN-24-NHS)

To the solution of compound 254b (26.3 mg, 0.028 mmol) in anhydrous 1,2dichloroethane (1 mL) was added trimethyltin hydroxide (74.6 mg, 0.413mmol). The mixture was heated to 80° C. and stirred overnight. It wascooled to room temperature, diluted with dichloromethane and washed withmixed solution of saturated sodium chloride and 5% hydrochloric acid (˜1mL), then brine. The organic layer was dried over anhydrous sodiumsulfate, filtered and evaporated. The residue was passed a short silicagel column and eluted with dichloromethane/methanol to remove the extratrimethyltin hydroxide. The product fractions were evaporated and highvacuumed to give the acid 254e as a yellowish solid (26 mg, yield=100%).MS (m/z): found 940.5 (M−H)⁻, 964.6 (M+Na)⁺. Compound 2542 (26 mg, 0.028mmol) was then dissolved in anhydrous dichloromethane (1 mL).N-hydroxysuccinimide (NHS, 9.57 mg, 0.083 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 15.9mg, 0.083 mmol) and DMAP (0.34 mg, 0.0028 mmol) was added subsequently.The mixture was stirred at room temperature overnight and diluted withdichloromethane, washed with saturated ammonium chloride and brine,dried over anhydrous sodium sulfate. It was filtered, evaporated and theresidue was purified by preparative reverse phase HPLC (C18 column,eluted with acetonitrile/water). The product fractions were combined andextracted with dichloromethane. The organic layers were dried overanhydrous sodium sulfate, filtered, evaporated and high vacuumed to givecompound 254f (IGN-24-NHS) as a light yellowish solid (3.0 mg,yield=10%). MS (m/z): found 1061.7 (M+Na)⁺. Note: DMAP should not havebeen added and it may be the cause of the low yield.

Example 22

Compound 255a

To a stirred solution of O-methyl-undecaethylene glycol (500 mg, 0.968mmol) in anhydrous dichloromethane (3 mL) was added triethylamine (270μl, 1.94 mmol), tosyl chloride (277 mg, 1.45 mmol) and DMAP (5.91 mg,0.048 mmol) subsequently at room temperature. The mixture continued tobe stirred overnight and worked up by diluted with ethyl acetate andfiltered to remove the triethylamine hydrochloride solid. The solid waswashed with ethyl acetate and the filtrate was evaporated. The residuewas diluted with ethyl acetate and filtered to remove the additionalprecipitate. The filtrate was evaporated to give the crude product asliquid. It was purified by silica gel chromatography(dichloromethane/methanol) to give compound 255a as a light yellowishoil (630 mg, yield=97%). ¹H NMR (400 Hz, CDCl₃): δ 7.81 (d, J=8.0 Hz,2H), 7.35 (d, J=8.0 Hz, 2H), 4.17 (t, J=4.8 Hz, 2H), 3.72-3.54 (m, 42H),3.39 (s, 3H), 2.46 (s, 3H); MS (m/z): found 693.6 (M+Na)⁺.

Compound 255b

To the mixture of the tosylate 255a (630 mg, 0.939 mmol) and aniline 28(238 mg, 0.939 mmol) in anhydrous DMF (3 mL) was added anhydrouspotassium carbonate (195 mg, 1.409 mmol) and potassium iodide (31.2 mg,0.188 mmol). The mixture was heated to 85° C. and stirred at thattemperature overnight. The solution was cooled to room temperature anddiluted with dichloromethane. It was filtered through celite and thesolid was washed with dichloromethane. The filtrate was evaporated andthe residue was diluted with dichloromethane and filtered again toremove the additional solid. The filtrate was evaporated and the residuewas purified by silica gel chromatography (hexanes/10% methanol in THF)to give compound 255b as a colorless oil (41.8 mg, yield=5.9%). ¹H NMR(400 Hz, CDCl₃): δ 6.66 (s, 2H), 6.65 (s, 1H), 4.60 (s, 4H), 3.69 (s,3H), 3.66-3.58 (m, 42H), 3.56-3.53 (m, 2H), 3.39-3.36 (m, 5H), 2.52(broad s, 2OH), 2.36 (t, J=7.2 Hz, 2H), 1.91 (p, J=7.2 Hz, 2H); ¹³C NMR(400 Hz, CDCl₃): δ 173.9, 148.5, 142.8, 113.4, 109.9, 72.1, 70.8, 70.7,68.9, 65.7, 59.2, 51.8, 50.9, 50.7, 31.3, 22.4; MS (m/z): found 774.3(M+Na)⁺.

Compound 255c (IGN-26-OMe)

To a stirred solution of compound 255b (41.8 mg, 0.056 mmol) inanhydrous dichloromethane (1 mL) was added triethylamine (27 μl, 0.196mmol). The mixture was cooled to −10° C. and methanesulfonyl chloride(12.9 μl, 0.167 mmol) was added slowly in 15 minutes. The solutioncontinued to be stirred between −10° C. to −5° C. for 60 minutes andquenched by addition of ice/water. It was diluted with ethyl acetate andwashed with cold water. The organic layer was dried over anhydroussodium sulfate, filtered, evaporated and high vacuumed to give themesylates as colorless oil. MS (m/z): found 930.3 (M+Na)⁺. The mesylates(30 mg, 0.033 mmol) was transferred to a 5 mL round bottom flask withethyl acetate, evaporated and high vacuumed. Compound 8 (29.2 mg, 0.099mmol) was added followed by addition of anhydrous DMF (0.5 mL),anhydrous potassium carbonate (22.8 mg, 0.165 mmol) and potassium iodide(5.5 mg, 0.033 mmol). The mixture was stirred at room temperatureovernight. It was diluted with dichloromethane and washed with brine.The organic layer was dried over anhydrous sodium sulfate, filtered andevaporated. The residue was purified by silica gel chromatography(hexanes/10% methanol in THF) to give 20.5 mg of a mixture whichcontained compound 255c. It was dissolved in anhydrous dichloromethane(0.3 mL). Triethylamine (4 μl, 0.03 mmol), tosyl chloride (3.8 mg, 0.02mmol) and DMAP (0.2 mg, 0.002 mmol) were added subsequently at roomtemperature. The mixture continued to be stirred at room temperatureovernight and then was evaporated. The residue was purified by silicagel chromatography (dichloromethane/methanol) to give 11 mg of lightyellowish foam. It was further purified by preparative reverse phaseHPLC (C18 column, eluted with CH₃CN/H₂O) to give compound 255c(IGN-26-OMe) as colorless foam (1.6 mg, yield=2.2%). MS (m/z): found1326.5 (M+Na)⁺, 1344.6 (M+H₂O+Na)⁺, 1362.5 (M+2H₂O+Na)⁺.

Example 23

Compound 256a

To a stirred solution of compound 7 (384 mg, 1.0 mmol) in absoluteethanol (6 mL) and anhydrous dichloromethane (2 mL) was added sodiumborohydride (37.8 mg, 1.0 mmol) at 0° C. After 30 minutes, the ice/waterbath was removed and the reaction mixture continued to be stirred atroom temperature overnight. The reaction was quenched by addition ofsaturated ammonium chloride and diluted with dichloromethane. Themixture was separated and the organic layer was washed with brine, driedover anhydrous sodium sulfate and filtered. The solvents were removedunder reduced pressure to give compound 256a as a white solid (369 mg,yield=96%). ¹H NMR (400 Hz, CDCl₃): δ 8.37 (d, J=8.0 Hz, 1H), 7.50 (s,1H), 7.40-7.24 (m, 6H), 7.18 (d, J=7.2 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H),6.12 (s, 1H), 5.06 (s, 2H), 4.40 (tt, J₁=10.0 Hz, J₂=3.6 Hz, 1H), 3.87(s, 3H), 3.52-3.41 (m, 3H), 2.78 (dd, J₁=16.8 Hz, J₂=3.6 Hz, 1H); ¹³CNMR (400 Hz, CDCl₃): δ 166.5, 152.1, 142.73, 142.70, 140.4, 136.3,129.5, 128.5, 127.9, 127.7, 127.1, 124.5, 123.8, 117.2, 114.5, 112.7,103.4, 70.5, 57.1, 56.2, 54.5, 33.1; MS (m/z): found 409.2 (M+Na)⁺.

Compound 256b

To a solution of compound 256a (369 mg, 0.955 mmol) in anhydrousacetonitrile (9 mL) was added iodomethane (65 μl, 1.05 mmol) andpotassium carbonate (158 mg, 1.15 mmol). The mixture was stirred, heatedto 82° C. and refluxed overnight. The reaction mixture was removed fromthe oil bath, cooled to room temperature and diluted withdichloromethane. It was filtered through celite and the filtrate wasevaporated under reduced pressure. The residue was purified throughsilica gel chromatography (hexanes/ethyl acetate) to give compound 256bas a colorless foam (195 mg, yield=51%). Also 123 mg of startingmaterial 256a was recovered. ¹H NMR (400 Hz, CDCl₃): δ 8.29 (d, J=8.0Hz, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.39-7.24 (m, 5H), 7.16 (d, J=7.2Hz, 1H), 7.04 (t, J=7.6 Hz, 1H), 6.46 (s, 1H), 5.19 (dd, J₁=15.2 Hz,J₂=12.4 Hz, 2H), 4.36-4.29 (m, 1H), 3.89 (s, 3H), 3.38-3.31 (m, 2H),3.02 (dd, J₁=10.8 Hz, J₂=4.0 Hz, 1H), 2.70 (dd, J₁=16.8 Hz, J₂=2.8 Hz,1H), 2.65 (s, 3H); ¹³C NMR (400 Hz, CDCl₃): δ 166.9, 151.2, 144.2,142.1, 141.9, 136.7, 129.8, 128.6, 128.0, 127.8, 127.3, 125.1, 123.9,121.7, 117.1, 113.5, 104.7, 71.1, 64.2, 57.2, 56.3, 40.2, 32.0; MS(m/z): found 423.2 (M+Na)⁺.

Compound 256c

To a stirred solution of compound 256b (195 mg, 0.487 mmol) in ethylacetate (2.5 mL) was added palladium on charcoal (10%, 25.9 mg, 0.024mmol). The flask was briefly vacuumed and replaced with H₂ in a balloon.The mixture continued to be hydrogenated for overnight and filteredthrough celite. The filtrate was stripped under reduce pressure and theresidue was purified by silica gel chromatography(dichloromethane/methanol) to give compound 256c as a white solid (126mg, yield=83%). ¹H NMR (400 Hz, MeOD): δ 8.14 (d, J=8.0 Hz, 1H),7.30-7.23 (m, 2H), 7.22 (s, 1H), 7.10 (t, J=7.6 Hz, 1H), 6.56 (s, 1H),4.46-4.38 (m, 1H), 3.88 (s, 3H), 3.48-3.37 (m, 2H), 3.12 (dd, J₁=10.8Hz, J₂=4.4 Hz, 1H), 2.84 (dd, J₁=16.8 Hz, J₂=2.8 Hz, 1H), 2.80 (s, 3H).

Compound 256d (IGN-29-OMe)

To a stirred solution of compound 249c (136 mg, 0.34 mmol) in anhydrousdichloromethane (2 mL) was added triethylamine (142 μl, 1.02 mmol). Themixture was cooled to −10° C. and methanesulfonyl chloride (66 μl, 0.85mmol) was added slowly in 15 minutes. The solution continued to bestirred between −10° C. to −5° C. for 60 minutes and quenched byaddition of ice/water. It was diluted with ethyl acetate and washed withcold water. The organic layer was dried over anhydrous sodium sulfate,filtered, evaporated and high vacuumed to give the mesylate as colorlessoil. The mesylate was transferred to a 10 mL round bottom flask withethyl acetate, evaporated and high vacuumed. Compound 8 (120 mg, 0.41mmol) and 256c (106 mg, 0.34 mmol) were added to it followed by additionof anhydrous DMF (1.5 mL), anhydrous potassium carbonate (235 mg, 1.7mmol). The mixture was stirred at room temperature overnight. It wasdiluted with dichloromethane and washed with brine. The organic layerwas dried over anhydrous sodium sulfate, filtered and evaporated. Theresidue was purified by preparative reverse phase HPLC (C18 column,eluted with CH₃CN/H₂O) to give compound 256d (IGN-29-OMe) as a lightyellowish solid (46 mg, yield=14%) and compound 256e. 256d: 1H NMR (400Hz, CDCl₃), δ 8.27 (d, J=8.0 Hz, 2H), 7.84 (d, J=4.8 Hz, 1H), 7.57 (s,1H), 7.32-7.04 (m, 7H), 6.87 (s, 1H), 6.82 (s, 1H), 6.76-6.70 (m, 2H),6.50 (s, 1H) 5.18-5.12)m, 4H), 4.49-4.43 (m, 1H), 4.40-4.35 (m, 1H),3.98 (s, 3H), 3.89 (s, 3H), 3.68-3.52 (m, 18H), 3.41-3.36 (m, 6H), 3.08(dd, J₁=10.8 Hz, J₂=4.4 Hz, 1H), 2.56 (dd, J₁=16.8 Hz, J₂=2.8 Hz, 1H),2.70 (s, 3H), 2.34 (t, J=7.2 Hz, 2H), 1.92-1.85 (m, 2H); MS (m/z): found990.6 (M+Na)⁺, 1008.6 (M+H₂O+Na)⁺. 256e: MS (m/z): found 1006.6 (M+Na)⁺.

Compound 256f and Compound 256g (IGN-29-NHS)

To the solution of compound 256d (46 mg, 0.048 mmol) in anhydrous 1,2dichloroethane (1.5 mL) was added trimethyltin hydroxide (129 mg, 0.71mmol). The mixture was heated to 80° C. and stirred overnight. It wascooled to room temperature, diluted with dichloromethane and washed withmixed solution of saturated sodium chloride and 5% hydrochloric acid (˜1mL), then brine. The organic layer was dried over anhydrous sodiumsulfate, filtered and evaporated. The residue was passed a short silicagel column and eluted with dichloromethane/methanol to remove the extratrimethyltin hydroxide. The product fractions were evaporated and highvacuumed to give the acid 256f as a yellowish solid (36.9 mg,yield=81%). MS (m/z): found 952.8 (M−H)⁻. Compound 256f (36.9 mg, 0.039mmol) was then dissolved in anhydrous dichloromethane (0.8 mL).N-hydroxysuccinimide (NHS, 13.4 mg, 0.12 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 22.2mg, 0.12 mmol) was added subsequently. The mixture was stirred at roomtemperature overnight and diluted with dichloromethane, washed withbrine and dried over anhydrous sodium sulfate. It was filtered,evaporated and the residue was purified by preparative reverse phaseHPLC (C18 column, eluted with acetonitrile/water). The fractionscontaining product were combined and extracted with dichloromethane. Theorganic layers were dried over anhydrous sodium sulfate, filtered,evaporated and high vacuumed to give compound 256g (IGN-29-NHS) as alight yellowish solid (25.4 mg, yield=62%). MS (m/z): found 1073.4(M+Na)⁺, 1091.4 (M+H₂O+Na)⁺, 1103.3 (M+3H₂O—H)⁻.

Example 24

methyl1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carboxylate(258b)

Methyl 6-nitroindoline-2-carboxylate (258a) (0.233 g, 1.048 mmol) wasdissolved in anhydrous tetrahydrofuran (4 ml) in a separate flask andcooled to 0° C. an ice bath. In another flask4-(benzyloxy)-5-methoxy-2-nitrobenzoyl chloride (4) (0.371 g, 1.153mmol) was dissolved in anhydrous tetrahydrofuran (4 ml) and cooled to 0°C. in an ice bath. To the flask containing the indoline was addedtriethylamine (0.438 ml, 3.15 mmol) via syringe and the acetyl chloride4 was added quickly to the reaction mixture via cannula at 0° C. Thereaction was stirred for 90 minutes at 0° C. and then at roomtemperature for an additional 1 hour. The reaction was then quenchedwith 5% HCl and extracted with ethyl acetate (3×). The combined organiclayers were washed with brine, dried over anhydrous sodium sulfate, andconcentrated in vacuo. The residue was purified by silica gelchromatography using 30% Acetone in hexane to give methyl1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carboxylate(258b) (0.220 g, 0.434 mmol, 41.4% yield) as a yellowish foam. ¹H NMR(400 Hz, CDCl₃): δ 3.30 (m, 1H), 3.60 (s, 3H), 3.69 (m, 1H), 3.86 (s,3H), 4.64 (dd, 1H, J=2.4 Hz, 10.8), 5.23 (s, 2H), 7.31 (m, 1H), 7.46 (m,6H), 7.99 (dd, 1H, J=2.0, 8.0 Hz), 9.04 (d, 1H, J=2.0 Hz). MS (m/z):found 530.1 ([M]⁺+Na).

1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carbaldehyde(258c)

Methyl1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carboxylate(258b) (1.023 g, 2.016 mmol) was dissolved in a mixture of anhydrousdichloromethane (2.5 mL) and toluene (7.5 mL) and cooled to −78° C. in adry ice and acetone bath. After 15 minutes DIBAL-H (1.0M in THF) (4.03mL, 4.03 mmol) was added via a syringe pump over about a 20 minuteperiod. The resulting solution was stirred for 2 hrs at −78° C. afterwhich methanol (1 ml) was added dropwise to quench the reaction at −78°C. The reaction was then diluted with 5% HCl and ethyl acetate andwarmed to room temperature. The aqueous layer was washed with additionalethyl acetate and the combined organic layers were washed with brine anddried over anhydrous sodium sulfate. The reaction mixture was passedthrough a layer of celite and concentrated in vacuo. The crude residuewas purified by silica gel chromatography using 40% acetone in hexane togive1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carbaldehyde(258c) (621 mg, 1.301 mmol, 64.5% yield). ¹H NMR (400 Hz, CDCl₃): δ3.15-3.60 (m, 2H), 3.90 (s, 0.6H), 3.92 (s, 1.2H), 3.97 (s, 1.2H), 4.57(d, 0.2H, J=4.8 Hz), 5.21 (m, 2.4H), 5.5 (m, 0.4H), 6.39 (s, 0.4H), 6.46(s, 0.2H), 6.76 (s, 0.2H), 6.89 (s, 0.4H), 7.01 (s, 0.4H), 7.19-7.41 (m,5.6H), 7.60-7.77 (m, 1.6H), 7.86-7.91 (m, 0.8H), 8.94 (s, 0.4H), 9.34(s, 0.4H), 9.74 (s, 0.4H), 9.90 (s, 0.2H). MS (m/z): found 500.1([M]⁺+Na).

Compound 258e

1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carbaldehyde (258c) (0.125 g, 0.262 mmol) was dissolved in tetrahydrofuran(8 mL) and water (5.33 mL). To this solution was added sodiumhydrosulfite (0.456 g, 2.62 mmol) and the reaction was capped with asepta and vented with a needle (no nitrogen/argon needed) and stirredovernight. Methanol was added to the reaction mixture and stirred anadditional 30 minutes at which point the reaction was concentrated invacuo to remove all solvents. The residue was dissolved in a 1:1 mixtureof methanol and dichloromethane (20 mL) which left a residue which didnot dissolve. The mixture was passed through a short pad of silica ontop of a short pad of celite and rinsed thoroughly with the 1:1 mixtureof methanol and dichloromethane. The filtrate was filtered again throughcelite and then a solution of HCl in dioxane (4M) was added withstirring until a pH of ˜3-4 was obtained. The reaction was then stirredfor an additional 30 minutes and then aqueous sodium bicarbonate wasadded until the reaction became basic (˜pH 8-9) at which time additionaldichloromethane was added and the organic layer removed. The aqueouslayer was washed with additional dichloromethane and the resultingorganic layers were combined and washed with brine, dried over anhydroussodium sulfate and concentrated in vacuo. The residue containingcompound 258d (0.105 g, 0.263 mmol, 100% yield) was used in the nextstep without further treatment. MS (m/z): found 454.2 ([M]⁺+Na+CH₃OH).

To a small vial containing 4-methyl-4-(methyldisulfanyl)pentanoic acid(0.061 g, 0.313 mmol), EDC (0.060 g, 0.313 mmol), and DMAP (0.038 g,0.313 mmol) were dissolved in dichloromethane (1 mL) with stirring. Tothis mixture compound 258d (0.125 g, 0.313 mmol) dissolved indichloromethane (1.5 mL) was added and the mixture was stirred at roomtemperature overnight. Water was added and the layers were separated.The organic layer was washed with brine, dried over anhydrous sodiumsulfate, and concentrated in vacuo. The residue was purified on a silicagel column using 50% ethyl acetate in hexane to give compound 258e(0.037 g, 0.064 mmol, 20.54% yield). ¹H NMR (400 Hz, CDCl₃): δ 1.27 (s,6H), 1.97 (t, 2H, J=8.0 Hz), 2.06 (t, 2H, J=8.0 Hz), 2.45 (s, 3H), 3.48(m, 1H), 3.67 (m, 1H), 3.99 (s, 3H), 4.49 (m, 1H), 5.24 (q, 2H, J=8.4Hz), 6.90 (s, 1H), 7.22 (d, 1H, J=8.0 Hz), 7.39 (m, 5H), 7.55 (s, 1H),7.82 (d, 1H, J=8.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 8.07 (s, 1H). MS (m/z):found 630.3 ([M]⁺+Na+MeOH).

Compound 258f

Compound 258e (0.0185 g, 0.032 mmol) was dissolved in anhydrousdichloromethane (0.5 ml) and to this solution was added methanesulfonicacid (0.021 ml, 0.321 mmol) dissolved in anhydrous dichloromethane(0.500 ml) and the resulting mixture was stirred at room temperature forthree hours. The reaction was poured over a mixture of ice and methanoland neutralized to pH 7 with aqueous sodium bicarbonate. The reactionwas then diluted with dichloromethane and the layers were separated. Theorganic layer was washed with brine, dried over anhydrous sodiumsulfate, and concentrated in vacuo. The residue was purified by silicaptlc using 3% methanol in dichloromethane to giveNH(4-methyl-4-methyldithio-pentanoate)-indole IGN monomer (0.007 g,0.014 mmol, 44.9% yield). MS (m/z): found 484.0 ([M]⁺−1).

Compound 258g

In a small vial dissolved Compound 8 (0.033 g, 0.112 mmol) in DMF (1.5ml) with stirring at room temperature. 1,3-diiodopropane (0.065 ml,0.561 mmol) was added followed by the addition of potassium carbonate(0.023 g, 0.168 mmol). The reaction was covered in foil and stirred atroom temperature overnight. The reaction was diluted withdichloromethane and washed with aqueous ammonium chloride and brine. Theorganic layer was dried over anhydrous sodium sulfate, filtered, andconcentrated in vacuo. The residue was purified by silica ptlc using 50%ethyl acetate in hexane to give Compound 258g (0.018 g, 0.039 mmol,34.7% yield). MS (m/z): found 533.0 ([M]⁺+K).

Compound 258h (IGN-15-SMe)

In a small vial dissolved Compound 258f (0.007 g, 0.014 mmol) indimethylformamide (1 ml) with stirring at room temperature. Compound 8(6.66 mg, 0.014 mmol) was added followed by the addition of potassiumcarbonate (1.992 mg, 0.014 mmol). The reaction was covered in foil andstirred at room temperature overnight. Reaction was diluted withdichloromethane and washed with aqueous ammonium chloride and brine. Theorganic layer was dried over anhydrous sodium sulfate, filtered, andconcentrated in vacuo. The residue was purified by silica ptlc plate in5% methanol in dichloromethane to give Compound 258h (IGN-15-SMe) (0.005g, 7.32 μmol, 50.8% yield). MS (m/z): found 906.3 ([M]⁺+Na+2CH3OH).

Example 25

Compound 259a

In a small vial dissolved Compound 8 (0.100 g, 0.340 mmol) in DMF (5 ml)with stirring at room temperature. 1,5-diiodopentane (0.506 ml, 3.40mmol) was added followed by the addition of potassium carbonate (0.070g, 0.510 mmol). The reaction was covered in foil and stirred at roomtemperature overnight. The reaction was diluted with dichloromethane andwashed with aqueous ammonium chloride and brine. The organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated invacuo. The residue was purified by silica ptlc using 50% ethyl acetatein hexane to give Compound 259a (0.045 g, 7.32 μmol, 27% yield). ¹H NMR(400 Hz, CDCl₃): δ 1.64 (m, 2H), 1.94 (M, 4H), 3.24 (t, 2H, J=6.5 Hz),3.52 (dd, 1H, J=4.0, 16.6 Hz), 3.73 (dd, 1H, J=10.5, 16.6 Hz), 3.98 (s,3H), 4.12 (m, 2H), 4.50 (dt, 1H, J=4.0, 11.2 Hz), 6.84 (s, 1H), 7.13 (t,1H, J=6.0 Hz), 7.29 (m, 2H), 7.57 (s, 1H), 7.90 (d, 1H, J=4.4 Hz), 8.29(d, 1H, J=8.0 Hz). MS (m/z): found 533.3 ([M]⁺+K).

Compound 259b (IGN-21-SMe)

In a small vial dissolved Compound 258f (15 mg, 0.031 mmol) indimethylformamide (1 ml) with stirring at room temperature. Compound259a (17.42 mg, 0.036 mmol) was added followed by the addition ofpotassium carbonate (4.27 mg, 0.031 mmol). The reaction was covered infoil and stirred at room temperature overnight. Reaction was dilutedwith dichloromethane and washed with aqueous ammonium chloride andbrine. The organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated in vacuo. The residue was purified by silicaptlc plate in 5% methanol in dichloromethane to give Compound 259a(IGN-15-SMe) (0.006 g, 7.32 μmol, 22% yield). MS (m/z): found 934.1([M]⁺+Na+2CH3OH).

Example 26

Compound 260a

Compound 256a (55 mg, 0.142 mmol) was dissolved in anhydrousdichloromethane and then 4-methoxy-4-oxobutanoic acid (76 mg, 0.575mmol), EDC (70 mg, 0.365 mmol), and DMAP (8.69 mg, 0.071 mmol) wereadded sequentially. The mixture was stirred overnight at roomtemperature and was checked by TLC to ensure no starting materialremained. The reaction was then diluted with water and ethyl acetate.After further extraction with ethyl acetate, the organic was washed withbrine, dried over sodium sulfate, filtered and concentrated in vacuo.The crude residue was purified by silica gel chromatography using 50%ethyl acetate in hexane to give compound 260a (54 mg, yield=76%). ¹H NMR(400 Hz, CDCl₃): δ 8.21 (d, J=8.0 Hz, 1H), 7.45-7.25 (m, 7H), 7.20 (d,J=7.2 Hz, 1H), 7.08 (t, J=7.4 Hz, 1H), 6.825 (s, 1H), 5.27 (q, J=15.1Hz, 2H), 4.56 (t, J=12.6 Hz, 1H), 4.35-4.29 (m, 1H), 3.99 (s, 3H), 3.65(s, 3H), 3.44-3.38 (m, 2H), 2.88 (dd, J₁=16.4 Hz, J₂=2 Hz, 1H),2.58-2.50 (m, 1H), 2.40-2.33 (m, 1H), 2.26-2.18 (m, 1H), 1.99-1.92 (m,1H); MS (m/z): found 523.1 (M+Na)⁺.

Compound 260b

To a solution of compound 260a (50 mg, 0.100 mmol) in anhydrousdichloromethane (11.5 ml) was added drop wise methanesulfonic acid(0.389 ml, 5.99 mmol) resulting in a yellow solution. The reactionstirred at room temperature and was monitored by TLC until completionbeginning at 30 minutes. It was diluted with water and methanol thenneutralized to pH 7 using saturated sodium bicarbonate. The aqueouslayer was extracted with dichloromethane and the organic layer driedover sodium sulfate. The crude product was purified by silica gelchromatography using 6% methanol in dichloromethane to give compound260b (40 mg, yield=98%). ¹H NMR (400 Hz, CDCl₃): δ 8.22 (d, J=8.0 Hz,1H), 7.35 (s, 1H), 7.28 (t, J=7.8 Hz, 1H), 7.22 (d, J=7.2 Hz, 1H), 7.09(t, J=7.4 Hz, 1H), 6.90 (s, 1H), 6.06 (s, 1H), 4.63 (t, J=12.6 Hz, 1H),4.38-4.30 (m, 1H), 4.00 (s, 3H), 3.66 (s, 3H), 3.47-3.39 (m, 2H), 2.90(dd, J₁=16.2 Hz, J₂=2.2 Hz, 1H), 2.69-2.59 (m, 2H), 2.52-2.45 (m, 1H),2.22-2.14 (m, 1H); MS (m/z): found 433 (M+Na)⁺.

Compound 260c

Compound 260b (20 mg, 0.049 mmol) and compound 259a (30 mg, 0.061 mmol)were dissolved in anhydrous N,N-dimethylformamide (1 ml). Potassiumcarbonate (20.20 mg, 0.146 mmol) was added and the reaction stirredovernight at room temperature. It was quenched with water and extractedwith dichloromethane. The organic was washed with brine and dried oversodium sulfate. The crude product was purified by silica gelchromatography using 5% methanol in dichloromethane to give compound260c (25 mg, yield=66%). MS (m/z): found 813.5 (M+Na+H₂O)⁺.

Example 27

Compound 261a

The commercially available starting material, thiazolidine-4-carboxylicacid (1.3 g, 9.59 mmol) was dissolved in anhydrous methanol (19.18 mL)and cooled to 0° C. in an ice bath. Thionyl chloride (1.40 mL, 19.18mmol) was added drop wise and the reaction stirred for 30 minutes. Theice bath was removed and stirring continued either for 4-5 hours orovernight. The solvent was stripped and the product placed on the highvacuum to give 4-(methoxycarbonyl)thiazolidin-3-ium chloride. Withoutfurther purification and assuming 100% yield, the4-(methoxycarbonyl)thiazolidin-3-ium chloride (1.761 g, 9.59 mmol) andcompound 4 (3.39 g, 10.55 mmol) were each dissolved separately intetrahydrofuran (32.0 mL) and cooled to 0° C. Triethylamine (4.41 mL,31.6 mmol) was added to the solution with4-(methoxycarbonyl)thiazolidin-3-ium chloride and then compound 4 wasadded quickly via canula. After 20 minutes, the pH of the solution waschecked to ensure it was basic. The reaction stirred at 0° C. for 1.5hours and then at room temperature for 30 minutes and was checked by MS.It was quenched with cold 5% hydrochloric acid and diluted with coldethyl acetate and water. The solution was extracted with ethyl acetatethree times and the combined organic washed with brine, saturated sodiumbicarbonate and then brine again. It was dried over sodium sulfate,filtered and stripped. The crude material was purified by silica gelchromatography using a gradient of 50% to 75% ethyl acetate in hexanesto give compound 261a (4.1 g, yield=99%). ¹H NMR (400 Hz, CDCl₃): thecompound appears as a pair of distinct rotomers. δ 7.78 (s, 0.6H), 7.74(s, 0.4H), 7.48-7.35 (m, 5H), 6.96 (s, 0.4H), 6.92 (s, 0.6H), 5.40 (dd,J₁=7.0 Hz, J₂=3.4 Hz, 0.6H), 5.31-5.22 (m, 2H), 5.13 (d, 9.6 Hz, 0.4H),4.60 (d, J=9.6 Hz, 0.4H), 4.46 (dd, J₁=4.4 Hz, J₂=3.2 Hz, 0.4H), 4.36(d, J=8.4 Hz, 0.6H), 4.26 (d, J=8.4 Hz, 0.6H), 4.02 (s, 1.8H), 3.96 (s,1.2H), 3.86 (s, 1.8H), 3.71 (s, 1.2H), 3.48-3.43 (m, 0.6H), 3.36-3.29(m, 1.4H); MS (m/z): found 455.3 (M+Na)⁺.

Compound 261b

Compound 261a (4.1 g, 9.48 mmol) was dissolved in dichloromethane (11mL) and toluene (33 mL) then cooled to −78° C. in an acetone/dry icebath. Diisobutylaluminium hydride (18.96 mL, 18.96 mmol) was added veryslowly, over at least 30 minutes, using a syringe pump. The reactionstirred at −78° C. for 3 hours and was quenched with methanol (0.4 mL)and then 5% hydrochloric acid (30 mL). Ethyl acetate (100 ml) was addedand the ice bath removed. The mixture continued to stir at roomtemperature for 30 minutes. It was extracted using ethyl acetate and thecombined organic washed with brine, saturated sodium bicarbonate, andthen brine again. It was dried over anhydrous sodium sulfate andfiltered through celite. The crude material was purified by silica gelchromatography using 75% ethyl acetate in hexanes to give compound 261b(2.3 g, yield=60%). ¹H NMR (400 Hz, CDCl₃): the compound appears as apair of rotomers. δ 9.80 (s, 0.8H), 9.41 (s, 0.2H), 7.80 (s, 0.8H), 7.73(s, 0.2H), 7.49-7.36 (m, 5H), 6.91 (s, 0.2H), 6.84 (s, 0.8H), 5.25-5.22(m, 2H), 4.85-4.73 (m, 1H), 4.35-4.30 (m, 1H), 4.22-4.17 (m, 1H),4.04-3.97 (m, 3H), 3.40-3.26 (m, 2H); MS (m/z): found 425.0 (M+Na)⁺.

Compound 261c

Compound 261b was dissolved in tetrahydrofuran (230 mL) then water (150mL). Sodium hydrosulfite (5.27 g, 25.7 mmol) was added slowly, in smallportions. If the solution remained cloudy, additional water was addeddrop wise until the solution cleared. The reaction was capped with asepta and needle to allow release of the SO₂ gas and was stirredovernight. The solution changed from a yellow to very pale, almostcolorless solution. The following morning, water was added until thesolution cleared and then methanol (30 mL) was added. It stirred for anadditional 2 hours and the solvents were then evaporated and the residuere-evaporated with acetonitrile at least twice. The white residue wasplaced on the high vacuum for a few hours. It was re-dissolved inmethanol: dichloromethane [1:1], filtered through celite, and stripped.The filter step was repeated until dilution in methanol appeared clearwith no particles. The intermediate was placed on the high vacuum untilcompletely dry then dissolved in anhydrous methanol (50 ml). Acetylchloride (1.9 ml, 26.7 mmol) was added drop wise at room temperature,causing a yellow precipitate to form. It stirred at room temperature for30 minutes and was quenched with saturated sodium bicarbonate. Themixture was diluted with dichloromethane and water (130 mL/85 mL) andextracted with dichloromethane. The aqueous layer was acidified withsodium hydrogensulfate, concentrated to a reduced volume, and thenre-extracted. The combined organic was washed with saturated sodiumbicarbonate and brine and dried over sodium sulfate. The strippedresidue was purified by silica gel chromatography using 60% ethylacetate in hexanes to give compound 261c (1.2 g, yield=59%). ¹H NMR (400Hz, CDCl₃): δ 7.69 (d, J=4.4 Hz, 1H), 7.52-7.28 (m, 6H), 6.87 (s, 1H),5.22 (q, J=12.3 Hz, 2H), 4.85, (d, J=10.4 Hz, 1H), 4.58 (d, J=10.4 Hz,1H), 4.03-4.02 (m, 1H), 3.98 (s, 3H), 3.51-3.47 (m, 1H), 3.45-3.23 (m,1H); MS (m/z): found 377.3 (M+Na)⁺.

Compound 261d

Compound 261c (75 mg, 0.212 mmol) was dissolved in neat trifluoroaceticacid (0.4 ml, 5.19 mmol). It refluxed for approximately 1 hour at 50° C.and then the temperature was increased to 80° C. After 3 hours total,the solvent was evaporated. The residue was directly purified by PTLCusing 5% methanol in dichloromethane to give compound 261d (19.4 mg,35%). ¹H NMR (400 Hz, CDCl₃): δ 7.72 (d, J=4.4 Hz, 1H), 7.51 (s, 1H),6.91 (s, 1H), 6.18 (s, 1H), 4.85 (d, J=10.4 Hz, 1H), 4.58 (J=10.4 Hz,1H), 4.05-4.02 (m, 1H), 3.99 (s, 3H), 3.50 (dd, J₁=12.4 Hz, J₂=6 Hz,1H), 3.32, (dd, J₁=12.4H, J₂=2 Hz, 1H); MS (m/z): found 319.0 (M+Na+MeOH)⁺.

Example 28

Compound 262

Compound 249c (18 mg, 0.045 mmol) was dissolved in anhydrousdichloromethane (0.45 mL) and then cooled in an ice/brine bath. First,triethylamine (0.022 ml, 0.158 mmol) and then methanesulfonyl chloride(10.46 μl, 0.135 mmol) were added; the second very slowly. The mixturecontinued to stir in the bath for 1 hour. The reaction was quenched withice/water and diluted with cold ethyl acetate. After separation, theorganic layer was washed again with cold water and dried over sodiumsulfate. It was filtered and evaporated under reduced pressure, keepingthe temperature below 20° C., and then placed on the high vacuum to beused directly. Once completely dry, the product, and compound 261d (28.5mg, 0.108 mmol) were dissolved in anhydrous N,N-dimethylformamide (350μL). Potassium carbonate (29.8 mg, 0.216 mmol) was added. After stirringovernight at room temperature, the reaction was diluted withdichloromethane, washed with brine, dried over sodium sulfate, filteredand stripped. The crude product was first purified by silica gelchromatography using 4% methanol in dichloromethane to remove baselineresidue. The recovered material was then purified using reverse phaseHPLC (C18 column, CH₃CN/H₂O, loaded column with 3:1, centrifuged beforeinjection) to give compound 262 as a solid. ¹H NMR (400 Hz, CDCl₃): δ7.68 (dd, J₁=4.4 Hz, J₂=1.6 Hz, 2H), 7.51 (s, 2H), 6.86 (s, 2H), 6.78(s, 1H), 6.71 (s, 2H), 5.16 (dq, J₁=8.4 Hz, J₂=2.2, 4H), 4.85 (d, J=10.4Hz, 2H), 4.58 (J=10.4 Hz, 2H), 4.04-3.97 (m, 7H), 3.68-3.38 (m, 18H),3.40-3.29 (m. 7H), 2.33 (t, 7.2 Hz, 2H), 1.89-1.35 (m, 2H) MS (m/z):found 914.1 (M+Na)⁺.

Example 29 IGN-13

methyl 3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)ethoxy)ethoxy)propanoate (263b)

To a stirred mixture of methyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (263a) (1.504 g,3.85 mmol) and (5-hydroxy-1,3-phenylene)dimethanol (21) (0.54 g, 3.50mmol) in anhydrous DMF (7.8 ml) was added potassium carbonate (0.726 g,5.25 mmol). The reaction was stirred at room temperature for 18 hours at75° C. The mixture was allowed to cool to room temperature, quenchedwith water, and extracted with ethyl acetate. The organic extracts werewashed with brine, dried over anhydrous magnesium sulfate, filtered andconcentrated. Purification by silica gel chromatography (5% MeOH/CH₂Cl₂)yielded methyl 3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)ethoxy)ethoxy)propanoate (263b)(340 mg, 26%). ¹H NMR (400 Hz, CDCl₃): δ6.83 (s, 1H), 6.75 (s, 2H), 4.52 (s, 4H), 4.05 (t, J=4.8 Hz, 2H), 3.79(t, J=4.8 Hz, 2H), 3.70 (t, J=6.4 Hz, 2H), 3.65 (s, 3H), 3.70-3.56 (m,8H), 3.26 (s, 2H), 2.55 (t, J=6.4 Hz, 2H); ¹³C NMR (400 Hz, CDCl₃): δ172.31, 159.1, 143.0, 117.7, 112.1, 70.8, 70.7, 70.5, 70.4, 69.8, 67.5,66.6, 64.7, 51.8, 34.9; MS (m/z): found 395.2 (M+Na)⁺.

Compound 263c

To a stirred solution of methyl3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)ethoxy)ethoxy)propanoate (263b) (145 mg, 0.389 mmol) in anhydrousdichloromethane (5.5 ml) was added triethylamine (0.163 ml, 1.168 mmol).The mixture was cooled to −5° C. and methanesulfonyl chloride (0.076 ml,0.973 mmol) was added slowly. After stirring for one hour at −5° C. thereaction was quenched with cold water and extracted with cold ethylacetate. The organic extracts were washed with cold water, dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give methyl 3-(2-(2-(2-(3,5-bis((methylsulfonyloxy)methyl)phenoxy)ethoxy)ethoxy)ethoxy) propanoate. MS (m/z): found 551.1 (M+Na)⁺.To a stirred mixture of methyl3-(2-(2-(2-(3,5-bis((methylsulfonyloxy)methyl)phenoxy)ethoxy)ethoxy)ethoxy)propanoate (206 mg, 0.390 mmol) andcompound 8 (287 mg, 0.974 mmol) in anhydrous DMF (3.9 ml) was addedpotassium carbonate (269 mg, 1.949 mmol). The reaction was allowed tostir at room temperature for 18 hours. The mixture was quenched withwater and extracted three times with dichloromethane. The organicextracts were washed with water and brine, dried over anhydrous sodiumsulfate, filtered and concentrated in vacuo. Purification by flashsilica gel chromatography (5% MeOH/CH₂Cl₂) followed by preparativereverse phase HPLC (C18 column, eluted with CH₃CN/H₂O) gave compound263c (110 mg, 30%) as a white solid. ¹H NMR (400 Hz, CDCl₃): δ 8.18 (d,J=8.0 Hz, 2H), 7.77 (m, 2H), 7.49 (s, 2H), 7.19 (m, 4H), 7.02 (m, 2H),6.89 (s, 2H), 6.87 (s, 1H), 6.75 (s, 2H), 5.10 (m, 4H), 4.39 (m, 2H),4.05 (m, 2H), 3.90 (s, 6H), 3.77 (m, 2H), 3.67 (t, J=6.4 Hz, 2H), 3.64(m, 2H), 3.59 (s, 3H), 3.70-3.54 (m, 8H), 3.40 (m, 2H), 2.51 (t, J=6.4Hz, 2H); MS (m/z): found 965.3 (M+H₂O+Na)⁺, 983.3 (M+2H₂O+Na)⁺.

Compound 263d

To a solution of compound 263c (51 mg, 0.055 mmol) in 1,2-Dichloroethane(2.2 ml) was added trimethyl tin hydroxide (199 mg, 1.103 mmol). Thereaction was stirred for 18 hours at 80° C., then cooled to roomtemperature, and quenched with saturated ammonium chloride. The mixturewas extracted with dichloromethane. The organic layer was washed withbrine, dried over anhydrous sodium sulfate, filtered and concentrated.Purification by silica gel chromatography (10% MeOH/CH₂Cl₂) yieldedcompound 263d (35 mg, 70%). ¹H NMR (400 Hz, CDCl₃): δ 8.26 (d, J=8.0 Hz,2H), 7.88 (m, 2H), 7.58 (s, 2H), 7.28 (m, 4H), 7.11 (m, 3H), 7.00 (s,2H), 6.88 (s, 2H), 5.21 (m, 4H), 4.49 (m, 2H), 4.18 (m, 2H), 4.00 (s,6H), 3.89 (m, 2H), 3.79 (m, 2H), 3.70 (m, 10H), 3.51 (m, 2H), 2.62 (m,2H); MS (m/z): found 909.2 (M−1)⁻, 927.2 (M−1+H₂O)⁻, 945.2 (M−1+2H₂O)⁻.

Compound 263e

To a solution of compound 263d (30 mg, 0.033 mmol) in anhydrousdichloromethane (2.5 mL) was added N-hydroxy succinimide (9.77 mg, 0.082mmol), EDC (15.78 mg, 0.082 mmol), and DMAP (0.406 mg, 3.29 μmol). Thereaction was stirred for 18 hours at room temperature and then dilutedwith dichloromethane. The mixture was washed with saturated ammoniumchloride and brine. The organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated in vacuo. The crude material waspurified by preparative reverse phase HPLC (C18 column, eluted withCH₃CN/H₂O). Fractions containing product were extracted withdichloromethane, dried over anhydrous sodium sulfate, filtered andco-evaporated with acetonitrile under reduced pressure to give compound263e (4.5 mg, 13%) as a white solid; MS (m/z): found 1030.4 (M+Na)⁺,1046.3 (M+K)⁺.

Example 30 IGN-27

methyl 3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenylamino)ethoxy)ethoxy)ethoxy)propanoate (264a)

To a mixture of methyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (263a) (250 mg,0.640 mmol) and (5-amino-1,3-phenylene)dimethanol (26)(108 mg, 0.704mmol) in anhydrous DMF (1.4 ml) was added potassium carbonate (133 mg,0.960 mmol). The reaction stirred for 18 hours at 80° C. and then wasallowed to cool to room temperature. The mixture was quenched with waterand extracted two times with ethyl acetate. The organic extracts werewashed with brine, dried over anhydrous magnesium sulfate, filtered andconcentrated. Purification by silica gel chromatography (5%Methanol/methylene chloride) yielded methyl3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenylamino)ethoxy) ethoxy)ethoxy)propanoate (264a) (61 mg, 25%); ¹H NMR (400 Hz, CDCl₃): δ 6.58(s, 1H), 6.47 (s, 2H), 4.49 (s, 4H), 3.67 (t, J=6.4 Hz, 2H), 3.62 (s,3H), 3.64-3.54 (m, 10H), 3.21 (t, J=5.2 Hz, 2H), 2.51 (t, J=6.4 Hz, 2H);MS (m/z): found 394.3 (M+Na)⁺.

Compound 264b

To a solution of methyl3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenylamino)ethoxy)ethoxy)ethoxy)propanoate (264a)(60 mg, 0.162 mmol) in acetonitrile (1.6ml) was added iodomethane (0.013 ml, 0.210 mmol) and potassium carbonate(26.8 mg, 0.194 mmol). The reaction stirred at 82° C. for 18 hours. Themixture was cooled to room temperature and then the solvent was removedunder reduced pressure. The crude material was diluted with 3:1CH₂Cl₂/MeOH and filtered through Celite. The filtrate was concentratedand purified by silica gel chromatography eluting with 5%Methanol/dichloromethane to give Compound 264b (35 mg, 56%). ¹H NMR (400Hz, CDCl₃): δ 6.58 (s, 3H), 4.52 (s, 4H), 3.64 (t, J=6.4 Hz, 2H), 3.60(s, 3H), 3.53 (m, 12H), 2.91 (s, 3H), 2.51 (t, J=6.4 Hz, 2H), 2.28 (s,2H); ¹³C NMR (400 Hz, CDCl₃): δ 172.1, 149.8, 142.4, 113.4, 109.9, 70.7,70.6, 70.4, 70.3, 68.6, 66.5, 65.6, 52.3, 51.7, 38.9, 34.8; MS (m/z):found 408.4 (M+Na)⁺.

Compound 264c

To a stirred solution of compound 246b (60 mg, 0.156 mmol) in anhydrousdichloromethane (2.8 mL) was added triethylamine (0.065 mL, 0.467 mmol).The mixture was cooled to −5° C. and methanesulfonyl chloride (0.030 mL,0.389 mmol) was added slowly. After stirring for one hour at −5° C. thereaction was quenched with cold water and extracted with cold ethylacetate. The organic layer was washed with cold water, dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give the dimesylate intermediate. MS (m/z): found 564.0(M+Na)⁺. To a mixture of the dimesylate linker (49 mg, 0.090 mmol) andcompound 8 (66.6 mg, 0.226 mmol) in anhydrous DMF (0.9 mL) was addedpotassium carbonate (62.5 mg, 0.452 mmol). The reaction was stirred for18 hours at room temperature, quenched reaction with water and extractedthree times with dichloromethane. The organic extracts were washed withwater and brine, dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo. Purification by flash silica gel chromatography(5% MeOH/CH₂Cl₂) followed by preparative reverse phase HPLC (C18 column,eluted with CH₃CN/H₂O) gave compound 264c (16 mg, 19%) as a white solid.¹H NMR (400 Hz, CDCl₃): δ 8.18 (d, J=8.0 Hz, 2H), 7.76 (m, 2H), 7.48 (s,2H), 7.18 (m, 4H), 7.02 (t, J=7.2 Hz, 2H), 6.79 (m, 2H), 6.74 (s, 1H),6.65 (s, 2H), 5.08 (m, 4H), 4.39 (m, 2H), 3.89 (s, 6H), 3.66 (t, J=6.4Hz, 2H), 3.62 (m, 2H), 3.60 (s, 3H), 3.53 (m, 12H), 3.40 (m, 2H), 2.91(s, 3H), 2.51 (t, J=6.4 Hz, 2H); MS (m/z), found 978.3 (M+H₂O+Na)⁺,996.3 (M+2H₂O+Na)⁺.

Compound 264d

To a solution of Compound 264c (26 mg, 0.028 mmol) in anhydrous1,2-Dichloroethane (1.1 ml) was added trimethyl tin hydroxide (100 mg,0.554 mmol). The reaction was stirred for 18 hours at 80° C. The mixturewas allowed to cool to room temperature and extracted withdichloromethane and saturated ammonium chloride. The organic extractswere washed with brine, dried over anhydrous sodium sulfate, filteredand concentrated in vacuo. Purification by preparative TLC in 5%Methanol/methylene chloride yielded compound 264d (14 mg, 55%). MS(m/z): found 922.1 (M−1)⁻, 940.0 (M−1+H₂O)⁻, 958.1 (M−1+2H₂O)⁻.

Compound 264e

To a stirred solution of compound 264d (13 mg, 0.014 mmol) in anhydrousdichloromethane (1.0 mL) was added N-hydroxysuccinimide (5.01 mg, 0.042mmol), EDC (8.09 mg, 0.042 mmol), and DMAP (0.172 mg, 1.407 μmol). Thereaction stirred for 18 hours at room temperature. The mixture wasextracted with dichloromethane and saturated ammonium chloride. Theorganic layer was washed with brine, dried over anhydrous sodiumsulfate, filtered and concentrated in vacuo. The crude material waspurified by preparative reverse phase HPLC (C18 column, eluted withCH₃CN/H₂O). Fractions containing product were combined and extractedwith dichloromethane, dried over anhydrous sodium sulfate, filtered andco-evaporated with acetonitrile under reduced pressure to obtaincompound 264e (4.1 mg, 29%). MS (m/z), found 1021.3 (M+H)⁺, 1043.2(M+Na)⁺, 1061.2 (M+H₂O+Na)⁺, 1079.2 (M+2H₂O+Na)⁺.

Example 31 IGN-28

methyl1-(tosyloxy)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265b)

To a stirred solution of methyl1-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265a)(1.2 g, 1.897 mmol) in dichloromethane (9.48 mL) at 0° C. wasadded triethylamine (0.529 mL, 3.79 mmol), toluene sulfonylchloride(0.542 g, 2.84 mmol) and DMAP (0.023 g, 0.190 mmol). The mixture wasstirred for one hour at 0° C. and then three hours at ambienttemperature, after which it was quenched with water and extracted twicewith dichloromethane. The organic extracts were washed with brine, driedover anhydrous magnesium sulfate, filtered and concentrated in vacuo.Purification by silica gel chromatography (5% MeOH/CH₂Cl₂) gave methyl1-(tosyloxy)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265b)(1.0 g, 67%) as a light yellow oil. ¹H NMR (400 Hz, CDCl₃): δ 7.80(d, J=8.4 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 4.16 (t, J=4.8 Hz, 2H), 3.75(t, J=6.4 Hz, 2H), 3.69 (s, 3H), 3.64 (m, 46H), 2.60 (t, J=6.4 Hz, 2H),2.45 (s, 3H).

methyl1-(3,5-bis(hydroxymethyl)phenylamino)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265c)

To a stirred mixture of methyl1-(tosyloxy)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265b)(700 mg, 0.890 mmol) and (5-amino-1,3-phenylene)dimethanol (26)(150 mg, 0.978 mmol) in anhydrous DMF (2.0 ml) was added potassiumcarbonate (184 mg, 1.334 mmol). The reaction was stirred at 80° C.overnight. The mixture was cooled to room temperature, quenched withwater and extracted with 10% Methanol/Methylene chloride. The organiclayer was washed with brine, dried over anhydrous magnesium sulfate,filtered and concentrated in vacuo. The crude product was purified bysilica gel chromatography (eluted with 5→15% MeOH/CH₂Cl₂) to give methyl1-(3,5-bis(hydroxymethyl)phenylamino)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265c) (285 mg, 42%). ¹H NMR (400 Hz, CDCl₃): δ 6.62 (s, 1H), 6.51 (s,2H), 4.52 (s, 4H), 3.72 (t, J=6.4 Hz, 2H), 3.65 (s, 3H), 3.61 (m, 48H),2.94 (s, 2H), 2.63 (s, 1H), 2.57 (t, J=6.4 Hz, 2H); MS (m/z): found790.4 (M+Na)⁺.

methyl2-(3,5-bis(hydroxymethyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oate(265d)

To a stirred solution of methyl1-(3,5-bis(hydroxymethyl)phenylamino)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate(265c) (67 mg, 0.087 mmol) in anhydrous DMF (1.0 ml) was addediodomethane (7.06 μl, 0.113 mmol) and potassium carbonate (14.47 mg,0.105 mmol). The reaction was stirred at 82° C. for 18 hours. Themixture was cooled to room temperature, diluted with water and extractedwith dichloromethane. The organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated in vacuo. Purification by preparativeTLC (10% MeOH/CH₂Cl₂) gave methyl2-(3,5-bis(hydroxymethyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oate(265d) (62 mg, 92%). ¹H NMR (400 Hz, CDCl₃): δ 6.65 (s, 3H), 4.59 (d,J=5.6 Hz, 4H), 3.74 (t, J=6.4 Hz, 2H), 3.67 (s, 3H), 3.61 (m, 46H), 3.54(t, J=6.0 Hz, 2H) 2.98 (s, 3H), 2.59 (t, J=6.4 Hz, 2H), 2.55 (m, 2H); MS(m/z): found 820.5 (M+K)⁺.

Compound 265e

To a stirred solution of methyl2-(3,5-bis(hydroxymethyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oate(265d) (71 mg, 0.091 mmol) in anhydrous dichloromethane (1.4 mL) wasadded triethylamine (0.038 mL, 0.272 mmol). The mixture was cooled to−5° C. and methanesulfonyl chloride (0.018 mL, 0.227 mmol) was addedslowly. After stirring for one hour at −5° C. the reaction was quenchedwith cold water and extracted with cold ethyl acetate. The organicextracts were washed with cold water, dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give methyl2-(3,5-bis((methylsulfonyloxy)methyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oate.MS (m/z): found 960.2 (M+Na)⁺. To a mixture of methyl2-(3,5-bis((methylsulfonyloxy)methyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oate(69 mg, 0.074 mmol) and compound 8 (54.1 mg, 0.184 mmol) in anhydrousDMF (0.8 mL) was added potassium carbonate (50.8 mg, 0.368 mmol). Thereaction was allowed to stir for 18 hours at room temperature. Thereaction was quenched with water and extracted twice withdichloromethane. The remaining aqueous layer was extracted twice with50% MeOH/CH₂Cl₂. The combined organic extracts were washed with brine,dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.Purification by flash silica gel chromatography (5% MeOH/CH₂Cl₂)followed by preparative reverse phase HPLC (C18 column, eluted withCH₃CN/H₂O) gave compound 265e (23 mg, 23%). MS (m/z): found 1375.4(M+Na+H₂O)⁺, 1393.4 (M+Na+2H₂O)⁺.

Compound 265f

To a stirred solution of compound 265e (22 mg, 0.016 mmol) in anhydrous1,2-dichloroethane (300 μL) was added trimethyl tin hydroxide (44.7 mg,0.247 mmol). The reaction stirred at 90° C. for 18 hours. The mixturewas allowed to cool to room temperature and then diluted withdichloromethane. The organic layer was washed with brine containing afew drops 5% concentrated hydrochloric acid and then with brine alone,dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.Purification by preparative TLC (2×5% MeOH/CH2Cl2) gave compound 265f(7.5 mg, 34%). MS (m/z): found 1318.4 (M−1)⁻, 1336.4 (M−1+H₂O)⁻, 1354.4(M−1+2H₂O)⁻.

Compound 265g

To a stirred solution of compound 265f (7.5 mg, 5.68 μmol) in anhydrousdichloromethane (400 μL) was added N-hydroxy succinimide (1.961 mg,0.017 mmol), EDC (3.27 mg, 0.017 mmol), and DMAP (0.069 mg, 0.568 μmol).The reaction stirred for 18 hours at room temperature. The mixture wasextracted with dichloromethane and saturated ammonium chloride. Theorganic layer was washed with brine, dried over anhydrous sodiumsulfate, filtered and the solvent was removed under reduced pressure.The crude material was purified by preparative reverse phase HPLC (C18column, eluted with CH₃CN/H₂O). Fractions containing product wereextracted with dichloromethane, dried over anhydrous sodium sulfate,filtered and co-evaporated with acetonitrile to give compound 265g (1.5mg, 19%). MS (m/z): found 1439.9 (M+Na)⁺, 1457.9 (M+Na+H₂O)⁺.

Example 32 IGN-22

Compound 266a

To a solution of compound 258d (20 mg, 0.050 mmol) in dichloromethane(1.0 mL) was added mono-methyl succinate (13.23 mg, 0.100 mmol), EDC(19.20 mg, 0.100 mmol), and DMAP (3.06 mg, 0.025 mmol) was added. Thereaction stirred at room temperature for 18 hours. The mixture wasdiluted with water and extracted with ethyl acetate. The organicextracts were washed with brine, filtered and concentrated under reducedpressure. Purification by silica gel chromatography (3% MeOH/CH₂Cl₂)gave compound 266a (15 mg, 58%). MS (m/z): found 568.4 (M+Na+MeOH)⁺.

Compound 266b

To a solution of compound 266a (15 mg, 0.029 mmol) in dichloromethane(3.5 ml) was added methanesulfonic acid (0.114 ml, 1.753 mmol). Thereaction was stirred for one hour at room temperature then diluted withmethanol and water. The mixture was neutralized with saturated sodiumbicarbonate to pH=7 and extracted three times with dichloromethane. Theorganic layer was dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo. Purification by preparative TLC (2×5%MeOH/CH₂Cl₂) gave compound 266b (11.5 mg, 93%). MS (m/z): found 446.4(M+Na)⁺, 478.4 (M+Na+ MeOH)⁺.

Compound 266c

To a mixture of compound 266b (11.5 mg, 0.027 mmol) and compound 259a(19.98 mg, 0.041 mmol) in anhydrous DMF (0.5 ml) was added potassiumcarbonate (11.26 mg, 0.081 mmol). The reaction was stirred for 18 hoursat room temperature. The mixture was quenched with water and extractedthree times with dichloromethane. The organic layer was washed withbrine, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure. Purification by preparative TLC (5% MeOH/CH₂Cl₂)followed by preparative reverse phase HPLC (C18 column, eluted withCH₃CN, H₂O) yielded compound 266c (4 mg, 18%). ¹H NMR (400 Hz, CDCl₃): δ8.27 (d, J=8.0 Hz, 1H), 8.06 (s, 1H), 7.87 (m, 2H), 7.74 (m, 1H), 7.55(s, 1H), 7.52 (s, 1H), 7.49 (m, 1H), 7.26 (m, 1H) 7.19 (d, J=8.8 Hz,1H), 7.10 (m, 1H), 6.82 (m, 2H), 4.49 (m, 2H), 4.12 (m, 4H), 3.95 (s,6H), 3.71 (s, 3H), 3.48 (m, 4H), 2.75 (m, 2H), 2.66 (m, 2H), 1.98 (m,4H), 1.70 (m, 2H); MS (m/z): found 824.1 (M+K)⁺.

Example 33 IGN-31

3,5-bis((tert-butyldimethylsilyloxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)aniline (267a)

To a solution of(5-(2-(2-(2-methoxyethoxy)ethoxy)ethylamino)-1,3-phenylene)dimethanol(249b)(0.4 g, 1.336 mmol) in dichloromethane (6.68 mL) was addedt-butyldimethylsilyl chloride (0.604 g, 4.01 mmol) and imidazole (0.318g, 4.68 mmol). The reaction stirred at room temperature for 90 minutes.The mixture was diluted with dichloromethane and filtered throughCelite. The filtrated was concentrated and purified by silica gelchromatography eluting with 20% Ethyl acetate/Hexanes to yield3,5-bis((tert-butyldimethylsilyloxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)aniline (267a) (600 mg, 85%). MS (m/z): found 550.3 (M+Na)⁺.

N-(3,5-bis((tert-butyldimethylsilyloxy)methyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide(267b)

To a mixture of3,5-bis((tert-butyldimethylsilyloxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)aniline(267a) (525 mg, 0.995 mmol) and 4-methyl-4-(methyldisulfanyl)pentanoicacid (232 mg, 1.193 mmol) in anhydrous dichloromethane (9.0 mL) wasadded EDC (229 mg, 1.193 mmol) and DMAP (12.15 mg, 0.099 mmol). Thereaction was stirred at room temperature for five hours. The mixture wasdiluted with dichloromethane and water. The organic layer was washedwith brine, dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. Purification by silica gelchromatography (30% Ethyl acetate/Hexanes) gaveN-(3,5-bis((tert-butyldimethylsilyloxy)methyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide(267b) (335 mg, 48%).

N-(3,5-bis(hydroxymethyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide(267c)

To a stirred solution ofN-(3,5-bis((tert-butyldimethylsilyloxy)methyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide(267b)(315 mg, 0.447 mmol) in anhydrous acetonitrile (7.0 mL) at 0° C.was added anhydrous pyridine (7.00 mL) followed by dropwise addition ofHF.Pyridine (3.1 mL, 1 mL/100 mg). The reaction stirred at 0° C. for twohours. It was diluted with ethyl acetate and slowly quenched withsaturated sodium bicarbonate. The mixture was extracted three times withethyl acetate. The organic layer was washed with water and brine, driedover sodium sulfate, filtered and concentrated. Purification by silicagel chromatography, eluting with 5% MeOH/CH₂Cl₂, yieldedN-(3,5-bis(hydroxymethyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide(267c)(190 mg, 89%). ¹H NMR (400 Hz, CDCl₃): δ 7.21 (s, 1H), 7.16 (s,2H), 4.63 (s, 4H), 3.79 (t, J=5.2, 5.6 Hz, 2H), 3.53 (m, 6H), 3.48 (m,4H), 3.29 (s, 3H), 2.53 (s, 2H), 2.27 (s, 3H), 2.07 (m, 2H), 1.84 (m,2H), 1.08 (s, 6H); MS (m/z): found 498.2 (M+Na)⁺.

Compound 267d

To a stirred solution ofN-(3,5-bis(hydroxymethyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide(267c) (72 mg, 0.151 mmol) in anhydrous dichloromethane (3.0 mL) wasadded triethylamine (0.063 mL, 0.454 mmol). The mixture was cooled to−5° C. and methanesulfonyl chloride (0.029 mL, 0.378 mmol) was addedslowly. After stirring for one hour at −5° C. the reaction was quenchedwith cold water and extracted with cold ethyl acetate. The organic layerwas washed with cold water, dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give(5-(N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamido)-1,3-phenylene)bis(methylene)dimethanesulfonate.MS (m/z): found 654.1 (M+Na)⁺. To a mixture of(5-(N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamido)-1,3-phenylene)bis(methylene)dimethanesulfonate(89 mg, 0.141 mmol) and compound 8 (83 mg, 0.282 mmol) in anhydrous DMF(1.5 mL) was added potassium carbonate (97 mg, 0.704 mmol). The reactionstirred for 18 hours at room temperature. The mixture was quenched withwater and extracted twice with dichloromethane. The organic layer waswashed with brine, dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo. Purification by silica gel chromatography (5%MeOH/CH₂Cl₂) and preparative reverse phase HPLC (C18 column, eluted withCH₃CN/H₂O) yielded compound 267d (27 mg, 18%). ¹H NMR (400 Hz, CDCl₃): δ8.28 (d, J=4.8 Hz, 2H), 7.87 (m, 2H), 7.61 (s, 2H), 7.37-7.27 (m, 7H),7.13 (t, J=7.2, 7.6 Hz, 2H), 6.88 (s, 2H), 5.25 (m, 4H), 4.50 (m, 2H),4.00 (s, 6H), 3.90 (m, 2H), 3.73 (m, 2H), 3.60 (m, 6H), 3.51 (m, 6H),3.30 (s, 3H), 2.32 (s, 3H), 2.15 (m, 2H), 1.90 (m, 2H), 1.13 (s, 6H); MS(m/z): found 1050.3 (M+Na)⁺, 1068.3 (M+H₂O+Na)⁺, 1086.3 (M+2H₂O+Na)⁺.

Example 34 IGN-32

Compound 268a

To a mixture of compound 253b (150 mg, 0.389 mmol) and tert-butyl3-(2-(2-(2-aminoacetamido)acetamido)acetamido)propanoate (148 mg, 0.467mmol) in anhydrous DMF (1.5 ml) was added EDC (90 mg, 0.467 mmol) andDMAP (4.75 mg, 0.039 mmol). The reaction stirred for 18 hours at roomtemperature. The mixture was directly purified by preparative reversephase HPLC (C18 column, eluted with CH₃CN/H₂O+0.1% formic acid). Furtherpurification by preparative TLC (15% MeOH/CH₂Cl₂) yielded compound 268a(170 mg, 64%). ¹H NMR (400 Hz, CDCl₃): δ 7.62 (m, 1H), 7.56 (m, 1H),7.38 (m, 1H), 7.11 (m, 1H), 6.55 (s, 2H), 6.52 (s, 1H), 4.45 (s, 4H),4.17 (s, 2H), 3.63 (m, 6H), 3.55-3.40 (m, 12H), 3.28 (m, 7H), 2.33 (t,J=6.4 Hz, 2H), 2.16 (m, 2H), 1.79 (m, 2H), 1.36 (s, 9H); MS (m/z): found706.3 (M+Na)⁺.

Compound 268b

To a stirred solution of compound 268a (59 mg, 0.086 mmol) in anhydrousdichloromethane (1.75 ml) was added triethylamine (0.036 ml, 0.259mmol). The mixture was cooled to −5° C. and methanesulfonyl chloride(0.017 ml, 0.216 mmol) was added slowly. After stirring for one hour at−5° C. the reaction was quenched with cold water and extracted with coldethyl acetate. The organic extracts were washed with cold water, driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure to give the desired dimesylate intermediate. MS (m/z): found862.3 (M+Na)⁺.

To a solution of the dimesylate intermediate (65 mg, 0.077 mmol) andcompound 8 (114 mg, 0.387 mmol) in anhydrous DMF (1.0 mL) was addedpotassium carbonate (86 mg, 0.619 mmol). The reaction was stirred for 18hours at room temperature, then quenched with water and extracted threetimes with dichloromethane. The organic layer was washed with brine,dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure. Purification by silica gel chromatography (2%→10%MeOH/CH₂Cl₂) yielded compound 268b (22 mg, 21%). ¹H NMR (400 Hz, CDCl₃):δ 8.26 (d, J=8.0 Hz, 2H), 7.88 (m, 2H), 7.58 (s, 2H), 7.28 (m, 4H), 7.13(t, J=7.2 Hz, 2H), 6.89 (s, 2H), 6.81 (s, 1H), 6.73 (s, 2H), 5.19 (m,4H), 4.48 m, 2H), 3.99 (s, 6H), 3.7-3.4 (m, 26H), 3.34 (s, 3H), 2.45 (t,J=6.4 Hz, 2H), 2.30 (m, 2H), 1.81 (m, 2H), 1.44 (s, 9H).

Example 35 Preparation of chB38.1-IGN14 Conjugate

A solution of chB38.1 antibody at a concentration of 2 mg/mL in anaqueous buffer containing 0.05 MN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) and 2 mMethylenediaminetetra-acetic acid (EDTA), pH 8 was treated with a 10-foldmolar excess of a solution of IGN14-NHS in dimethylacetamide (DMA) suchthat the final concentration of DMA in the buffer was 10% v/v. Thereaction mixture was stirred at room temperature for 120 min and thenloaded onto a Sephadex G25 gel filtration column (HiPrep™ 26/10Desalting Column GE#17-5087-01) that had been previously equilibratedinto an aqueous buffer containing 10 mM histidine, 250 mM glycine, 1%sucrose pH 5.5. The conjugated antibody-containing fractions werecollected and pooled to yield product. The pooled sample was dialyzedovernight against the same elution buffer to further purify the product.The final conjugate was assayed spectrophotometrically using theextinction coefficients that were determined for IGN-14 (ε₃₃₀=15,231 M⁻¹cm⁻¹ and ε₂₈₀=26,864 M⁻¹ cm⁻¹) and chB38.1 antibody (ε_(280 nm)=204,000M⁻¹cm⁻¹). An average of 3.3 IGN14 molecules per molecule of antibodywere linked.

Example 36 Preparation of huMy9-6-IGN23 Conjugate

A solution of huMy9-6 antibody at a concentration of 2 mg/mL in anaqueous buffer containing 0.05 MN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) and 2 mMethylenediaminetetra-acetic acid (EDTA), pH 8.5 was treated with a12.5-fold molar excess of a solution of IGN23-NHS in dimethylacetamide(DMA), glycerol, and sucrose. The final concentration of DMA, glyceroland sucrose in the buffer was 15%, 5% and 5% (v/v) respectively. Thereaction mixture was stirred at room temperature for 120 min and thenloaded onto a Sephadex G25 gel filtration column (HiPrep™ 26/10Desalting Column GE#17-5087-01) that had been previously equilibratedinto an aqueous buffer containing 10 mM histidine, 250 mM glycine, 1%sucrose, pH 5.5. The conjugated antibody-containing fractions werecollected and pooled to yield product. The pooled sample wasconcentrated using Millipore centrifugal filter devices, and thendialyzed overnight against the same elution buffer to further purify theproduct.

The final conjugate was assayed spectrophotometrically using theextinction coefficients that were determined for IGN-23 (e₃₃₀=15,231 M⁻¹cm⁻¹ and e₂₈₀=26,864 M⁻¹ cm⁻¹) and huMy9-6 (e_(280 nm)=206,460 M-1cm-1).An average of 2.2 IGN23 molecules per molecule of antibody were linked.

Example 37 Preparation of chB38.1-IGN27 Conjugate

A solution of chB38.1 antibody at a concentration of 2 mg/mL in anaqueous buffer containing 0.05 MN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) and 2 mMethylenediaminetetra-acetic acid (EDTA), pH 8.5 was treated with a12-fold molar excess of a solution of IGN27-NHS in dimethylacetamide(DMA, 5 mM stock) such that the final concentration of DMA in the bufferwas 15% v/v. The reaction mixture was stirred at room temperature for 4hr and then loaded on to a Sephadex G25 gel filtration column (HiPrep™26/10 Desalting Column GE#17-5087-01) that had been previouslyequilibrated into an aqueous buffer containing PBS pH 7.4. Theconjugated antibody-containing fractions were collected and pooled toyield product. The pooled sample was dialyzed overnight against the sameelution buffer to further purify the product.

The final conjugate was assayed spectrophotometrically using theextinction coefficients that were determined for IGN-27 (e₃₃₀=15,231 M⁻¹cm⁻¹ and e₂₈₀=26,864 M⁻¹ cm⁻¹) and chB38.1 antibody (e_(280 nm)=204,000M-1cm-1). An average of 2.9 IGN27 molecules per molecule of antibodywere linked.

Example 38 In Vitro Potency IGN Free Drugs and IGN Conjugates

General Procedure Used: Samples of IGN Free Drugs or IGN Conjugates wereadded to 96-well flat bottomed tissue culture plates and titrated usingserial dilutions to cover the desired molar range. Antigen positive(Ag+) or Antigen negative (Ag−) cells were added to the wells inspecific cell densities in such a way that there were triplicate samplesfor each drug concentration for each corresponding cell line. The plateswere then incubated at 37° C. in an atmosphere of 5% CO₂ for 4-5 daysdepending on the cell line. COLO 205 (1,000 cells/well), Namalwa (3,000cells/well)—4 days; RH30 (1,000 cells/well), Ramos (10,000 cells/well),KB (1,000 cells/well)—5 days)

At the end of the incubation period cytotoxic potencies were thenassessed using a WST-based cell viability assay and surviving cells weremeasured by developing with WST (2-7 hours). The absorbance in each wellwas measured and the surviving fraction of cells at each concentrationwas plotted to reveal the cytotoxicity and antigen specificity (of theconjugates).

The cytotoxicity of the IGN Free Drugs and the potency and specificityof the IGN conjugates were measured against a panel of human cancer celllines selected from COLO 205, NB-4, LOVO, Namalwa, RH30, Ramos, KB,and/or LOVO. Results are illustrated in FIGS. 51-58.

FIG. 51: Table which demonstrates the high potency (in nM) of the IGNFree Drugs against multiple cell lines. In general the IGN Free Drugsare found to be potent in the low picomolar range against this panel ofcell lines.

FIG. 52: (A) chB38.1-IGN13 conjugate (3.8 IGN/Ab) was found to be potentat sub-picomolar levels against COLO 205 (Ag+) cells and the activitywas significantly diminished (0.26 nM) when the antigen binding siteswere blocked with 1 μM unconjugated chB38.1 antibody indicating the highspecificity of this conjugate (>260 fold). (B) chB38.1-IGN13 conjugate(3.8 IGN/Ab) was found to be potent picomolar levels (0.002 pM) againstLOVO (Ag+) cells in a clonogenic assay.

FIG. 53: huMy9-6-IGN13 conjugate (3.4 IGN/Ab) was found to be potent atpicomolar levels against NB-4 (Ag+) cells (0.077 nM) and the activitywas significantly diminished (1.0 nM) when the antigen binding siteswere blocked with 1 μM huMy9-6 antibody indicating that this conjugateis specific.

FIG. 54: (A) chB38.1-IGN14 conjugate (3.1 IGN/Ab) was found to be potentat sub-picomolar levels against COLO 205 (Ag+) cells and the activitywas significantly less towards Namalwa (Ag−) cells (0.9 nM) indicatingthe high specificity of this conjugate (>900 fold). (B) chB38.1-IGN14conjugate (2.6 IGN/Ab) was found to be very potent towards LOVO (Ag+)cells (0.012 nM) and the activity was significantly less towards Namalwa(Ag−) cells (>3.0 nM) indicating the high specificity of this conjugate(>250 fold).

FIG. 55: huMy9-6-IGN14 conjugate (3.3 IGN/Ab) was found to be highlypotent against NB-4 (Ag+) cells (0.033 nM) and the activity wassignificantly less towards Namalwa (Ag−) cells (0.6 nM) indicating thehigh specificity of this conjugate.

FIG. 56: (A) chB38.1-IGN23 conjugate (2.5 IGN/Ab) was found to be potentat picomolar levels against LOVO (Ag+) cells (0.063 nM) and the activitywas significantly less towards Namalwa (Ag−) cells (>3.0 nM) indicatingthe high specificity of this conjugate. (B) chB38.1-IGN23 conjugate (2.0IGN/Ab) was found to be potent at picomolar levels against COLO 205(Ag+) cells (0.006 nM) and the activity was significantly diminished(2.5 nM) when the antigen binding sites were blocked with 1 μM chB38.1indicating that this conjugate is specific.

FIG. 57: chB38.1-IGN29 conjugate (2.8 IGN/Ab) was found to be potent atsub-nanomolar levels against COLO 205 (Ag+) cells (0.410 nM) and theactivity was significantly diminished (18 nM) when the antigen bindingsites were blocked with 1 μM chB38.1 indicating that this conjugate isspecific.

Example 39 In Vivo Efficacy of chB38.1-IGN14 Conjugate in COLO 205 TumorBearing Nude Mice

In this study, the anti-tumor activity of chB38.1-IGN14 was investigatedin female nude mice bearing COLO 205 tumors, a human colon carcinomamodel. COLO 205 tumor cells, 2×10⁶ cells/mouse were subcutaneouslyinoculated at a volume of 0.1 mL/mouse in the area over the rightshoulder of female athymic nude mice, 5 weeks of age. Eight days aftertumor cell inoculation mice were randomized into groups (n=6 per group)by tumor volume. Treatment was initiated the day of randomization, andgroups included a control group dosed with PBS (200 μL/injection), nakedchB38.1 antibody (2.8 mg/kg), non-targeting chKTI-IGN14 (50 μg/kg)conjugate and chB38.1-IGN14 (50 μg/kg IGN14 dose; 2.5 mg/kg antibodydose). All treatments were administered twice on a weekly schedule (day8 and 15, post-cell inoculation). Arrows indicate dosing times postinoculation. All treatments were well tolerated with the mean bodyweight losses comparable to loss seen in PBS control mice. Median tumorvolume vs time is shown (FIG. 58) with the data demonstrating theanti-tumor activity of the chB38.1-IGN14 conjugate. Both thenon-targeting and the naked antibody show no activity beyond that seenwith the vehicle control, suggesting that the anti-tumor activityobserved with the chB38.1-IGN-14 conjugate is antigen-specific.

Example 40

FIG. 59 shows the mass spectrum of chB38.1-IGN14 (deglycosylatedantibody). Peaks are labeled D1-D7 to indicate the number of IGN14molecules attached per antibody. The average number of IGN14 moleculesper antibody was calculated to be 3.5 (matching drug load calculated byUV-vis).

We claim:
 1. A conjugate comprising a cell binding agent (CBA)covalently linked, through a linking group, to a compound represented byformula (IV) or (V):

or a pharmaceutically acceptable salt, optical isomer, racemate,diastereomer, or enantiomer of the compound, wherein: the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is —H, and when it is asingle bond, X is —H or an amine protecting moiety that is capable ofbeing enzymatically or hydrolytically cleaved; Y is selected from thegroup consisting of —OR, —OCOR′, —OCOOR′, —OCONR′R″, —NR′R″, —NRCOR′,—NRCOP, wherein P is an amino acid or a polypeptide containing between 2to 20 amino acid units, —SR′, —SOR′, —SO₂R′, —SO₃, —OSO₃, a halogen, acyano, and an azido, wherein R, R′ and R″ are the same or different andare selected from the group consisting of —H, an optionally substitutedlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10carbon atoms, a polyethylene glycol unit (—OH₂CH₂)_(n), wherein n is aninteger from 1 to 2000, a 5- or 6-membered heteroaryl ring containingone or more heteroatoms independently selected from the group consistingof nitrogen, oxygen, and sulfur, a 5- to 18-membered fused ring system,wherein at least one of the rings is aromatic, containing one or moreheteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur, an aryl having from 6 to 18 carbon atoms,and a 3- to 18-membered heterocyclic ring having 1 to 6 heteroatomsselected from the group consisting of O, S, N and P, wherein theoptional substituent is selected from the group consisting of halogen,—OR₇, —NR₈R₉, —NO₂, —NRCOR′, —SR₁₀, —SOR′, —SO₂R′, —SO₃, —OSO₃,—SO₂NRR′, a cyano, an azido, —COR₁₁, —OCOR₁₁ or —OCONR₁₁R₁₂, wherein R₇,R₈, R₉, R₁₀, R₁₁ and R₁₂ are each independently selected from the groupconsisting of —H, a linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, a 5- or6-membered heteroaryl ring containing one or more heteroatomsindependently selected from the group consisting of nitrogen, oxygen,and sulfur, a 5- to 18-membered fused ring system, wherein at least oneof the rings is aromatic, containing one or more heteroatomsindependently selected from the group consisting of nitrogen, oxygen,and sulfur, an aryl having from 6 to 18 carbon atoms, and a 3- to10-membered heterocyclic ring having 1 to 6 heteroatoms selected from O,S, N and P, and R₁₀ is —SR₁₃ or —COR₁₃, wherein R₁₃ is selected from thegroup consisting of a linear, branched or cyclic alkyl, alkenyl oralkynyl having from 1 to 10 carbon atoms, a 5- or 6-membered heteroarylring containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, a 5- to 18-membered fused ring system,wherein at least one of the rings is aromatic, containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur, anaryl having from 6 to 18 carbon atoms, and a 3- to 18-memberedheterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P,optionally R₁₁ is —OR₁₄, wherein R₁₄ has the same definition as R,optionally R″ is —OH; W is C═O; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′are each independently selected from the group consisting of —H, anoptionally substituted linear, branched or cyclic alkyl, alkenyl oralkynyl having from 1 to 10 carbon atoms, and a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000, wherein theoptional substituent is selected from the group consisting of halogen,—NH(C═NH)NH₂, —OR₇, —NR₈R₉, —NO₂, —NRCOR′, —SR₁₀, —SOR′, —SO₂R′, —SO₃,—OSO₃, —SO₂NRR′, a cyano, an azido, —COR₁₁, —OCOR₁₁, —OCONR₁₁R₁₂, and apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000, optionally, any one of R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, or R₄′ iscovalently linked, through the linking group, to the cell binding agentor is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl,polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolylunit optionally linked to the linking group; Z is CH₂; R₆ is —OR, —SR or—NRR′, optionally R₆ is linked to the linking group; A and A′ are thesame or different and are selected from O, CRR′O, S, CRR′S, NR₁₅ orCRR′NHR₁₅, wherein R₁₅ has the same definition as R; D and D′ are thesame or different and are independently a linear, branched or cyclicalkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, optionallysubstituted with any one of halogen, —OR₇, —NR₈R₉, —NO₂, —NRCOR′, —SR₁₀,—SOR′, —SO₂R′, —SO₃, —OSO₃, —SO₂NRR′, a cyano, an azido, —COR₁₁, —OCOR₁₁or —OCONR₁₁R₁₂, and a polyethylene glycol unit (—OCH₂CH₂)_(n), wherein nis an integer from 1 to 2000; L is absent, or is a phenyl group or 3- to18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O,S, N and P, wherein said phenyl group or 3- to 18-membered heterocyclicring represented by L is optionally linked to the linking group, or isselected from a linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, optionally substituted with any one ofhalogen, —OR₇, —NR₈R₉, —NO₂, —NRCOR′, —SR₁₀, —SOR′, —SO₂R′, —SO₃, —OSO₃,—SO₂NRR′, a cyano, an azido, —COR₁₁, —OCOR₁₁ —OCONR₁₁R₁₂, and apolyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is an integer from 1to 2000; optionally, L itself is the linking group; provided that theconjugate has no more than one linking group.
 2. The conjugate of claim1, wherein: Y is selected from the group consisting of —OR, —NR′R″,—SO₃, and —OSO₃, wherein R is selected from the group consisting of —H,a linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to10 carbon atoms, a polyethylene glycol unit (—OCH₂CH₂)_(n), wherein n isan integer from 1 to 2000, an aryl having from 6 to 10 carbon atoms, anda heterocyclic ring having from 3 to 10 carbon atoms; W is C═O; R₁, R₂,R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently selected from —H,—NO₂ or the linking group; R₆ is —OR₁₈, wherein R₁₈ has the samedefinition as R; Z is CH₂; A and A′ are each O; D and D′ are the same ordifferent and are independently a linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms; L is absent, or isa phenyl group or a heterocyclic ring having from 3 to 10 carbon atoms,wherein said phenyl group or heterocyclic ring represented by L isoptionally linked to the linking group, or is a linear, branched orcyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms,optionally substituted with any one of halogen, —OR₇, —NR₈R₉, —NO₂,—NRCOR′, —SR₁₀, —SOR′, —SO₂R′, —SO₃, —OSO₃, —SO₂NRR′, a cyano, an azido,—COR₁₁, —OCOR₁₁ or and —OCONR₁₁R₁₂, or a polyethylene glycol unit(—OCH₂CH₂)_(n), wherein n is an integer from 1 to 2000; optionally, Litself is the linking group.
 3. The conjugate of claim 1, selected fromformulae (XII) and (XIII):

or a pharmaceutically acceptable salt, optical isomer, racemate,diastereomer, or enantiomer thereof, wherein Y is selected from thegroup consisting of —OH, —OR, —NR′R″, —SO₃, and —OSO₃, wherein R, R′ andR″ are a linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms; one of R₂, R₃, R₂′ and R₃′ is the linkinggroup and the others are —H or —NO₂; R₆ is —OR; Z is CH₂; A is O orNR₁₅; L is (CH₂)_(nn), wherein nn is 0 or an integer between 1 and 5, ora substituted or unsubstituted alkyl or alkenyl having from 2 to 4carbon atoms, wherein the substituent is selected from halogen, —OR₇,—NR₈R₉, —NO₂, —NRCOR′, —SR₁₀, —SOR′, —SO₂R′, —SO₃, —OSO₃, —SO₂NRR′, acyano, an azido, —COR₁₁, —OCOR₁₁ or —OCONR₁₁R₁₂, optionally, L itself isthe linking group; one of L′, L″ or L′″ is the linking group, while theothers are —H; preferably L′ is the linking group; and G is CH or N. 4.The conjugate of claim 1, selected from formulae (XIV) and (XV):

or a pharmaceutically acceptable salt, optical isomer, racemate,diastereomer, or enantiomer thereof, wherein Y is selected from thegroup consisting of —OH, —OR, —SO₃, and —OSO₃, wherein R is a linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms; nn is 0 or an integer from 1 to 5; one of R₂, R₃, R₂′ and R₃′ informula (XIV) is the linking group and the others are —H or —NO₂; one ofL′, L″ or L′″ in formula (XV) is the linking group, and the others are—H; and G is CH or N.
 5. The conjugate of claim 1, wherein one of R₁,R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, L′, L″, L′″ is covalently linked to theCBA, and is selected from the group consisting of:—O(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉N—NR₃₀)_(n″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,and—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,wherein: m, n, p, q, m′, n′, t′ are integer from 1 to 10, or areoptionally 0; m″, n″ and p″ are 0 or 1; X″ is covalently linked to theCBA, and when Y″ is not S—S and t=0, X″ is

—CH₂—CO—, or —S—; and when t=1, X″ is —NH—; Y″ is absent or is selectedfrom O, S, S—S or NR₃₂, wherein R₃₂ has the same definition as givenabove for R; A″ is an amino acid selected from glycine, alanine,leucine, valine, lysine, citrulline, glutamate, or a polypeptidecontaining between 2 to 20 amino acid units; R₂₀, R₂₁, R₂₂, R₂₃, R₂₄,R₂₅, R₂₆, and R₂₇ are the same or different and are —H or a linear orbranched alkyl having from 1 to 5 carbon atoms; R₂₉ and R₃₀ are the sameor different and are —H or an alkyl from 1 to 5 carbon atoms; R₃₃ is —Hor a linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1to 12 carbon atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n), or R₃₃ is—COR₃₄, —CSR₃₄, —SOR₃₄, or —SO₂R₃₄, wherein R₃₄ is —H or linear, abranched or cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbonatoms or, a polyethylene glycol unit —(OCH₂CH₂)_(n); and one of R₄₀ andR₄₁ is optionally a negatively or positively charged functional groupand the other is —H or an alkyl, alkenyl, alkynyl having 1 to 4 carbonatoms.
 6. The conjugate of claim 1, wherein the cell-binding agent bindsto target cells selected from tumor cells, virus infected cells,microorganism infected cells, parasite infected cells, autoimmune cells,activated cells, myeloid cells, activated T-cells, B cells, ormelanocytes; cells expressing the CD4, CD6, CD19, CD20, CD22, CD30,CD33, CD37, CD38, CD40, CD44, CD56, EpCAM, CanAg, CALLA, or Her-2antigens; Her-3 antigens or cells expressing insulin growth factorreceptor, epidermal growth factor receptor, and folate receptor.
 7. Theconjugate of claim 6, wherein the tumor cells are selected from breastcancer cells, prostate cancer cells, ovarian cancer cells, colorectalcancer cells, gastric cancer cells, squamous cancer cells, small-celllung cancer cells, and testicular cancer cells.
 8. The conjugate ofclaim 1, wherein the cell-binding agent is an antibody, a single chainantibody, an antibody fragment that specifically binds to the targetcell, a monoclonal antibody, a single chain monoclonal antibody, or amonoclonal antibody fragment that specifically binds the a target cell,a chimeric antibody, a chimeric antibody fragment that specificallybinds to the target cell, a domain antibody, a domain antibody fragmentthat specifically binds to the target cell, a lymphokine, a hormone, avitamin, a growth factor, a colony stimulating factor, or anutrient-transport molecule.
 9. The conjugate of claim 8, wherein theantibody is a resurfaced antibody, a resurfaced single chain antibody,or a resurfaced antibody fragment.
 10. The conjugate of claim 8, whereinthe antibody is a monoclonal antibody, a single chain monoclonalantibody, or a monoclonal antibody fragment thereof.
 11. The conjugateof claim 8, wherein the antibody is a chimeric antibody, a chimericantibody fragment, a domain antibody, or a domain antibody fragment. 12.The conjugate of claim 8, wherein the antibody is a humanized antibody,a humanized single chain antibody, or a humanized antibody fragment. 13.The conjugate of claim 8, wherein the antibody is humanized orresurfaced MY9, humanized or resurfaced anti-B4, or humanized orresurfaced C242.
 14. The conjugate of claim 1, wherein the compound isselected from the group consisting of:

wherein the wavy line represents a linkage to the cell-binding agent.15. A pharmaceutical composition comprising a conjugate of claim 1, anda pharmaceutically acceptable carrier.
 16. The pharmaceuticalcomposition of claim 15, further comprising a second compound, which isa chemotherapeutic agent.
 17. A method of treating a cancer in a mammal,comprising administering to said mammal a therapeutically effectiveamount of the conjugate of claim 1, wherein the cancer is selected fromthe group consisting of lymphoma, leukemia, colon cancer, cervicalcancer, epidermoid carcinoma, sarcoma, breast cancer, multiple myeloma,and melanoma.
 18. The method of claim 17, further comprisingadministering to said mammal a second compound, which is achemotherapeutic agent.
 19. The method of claim 18, wherein saidchemotherapeutic agent is administered to said mammal sequentially. 20.The method of claim 17, wherein the proliferative disorder is Burkitt'slymphoma, colon cancer, rhabdomyosarcoma, acute promyelocytic leukemia,cervical cancer, epidermoid carcinoma, multiple myeloma, breast cancer,acute lymphoblastic leukemia (ALL), or melanoma.
 21. The method of claim20, wherein the colon cancer is a multi-drug resistant (MDR) coloncancer.
 22. The method of claim 20, wherein the Burkitt's lymphoma is amulti-drug resistant (MDR) Burkitt's lymphoma.